ISOLATION AND CHARACTERIZATION OF

VIRUS-LIKE PARTICLES FROM

______

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

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! 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 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 . 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 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 VLP isolates from 04/13/12...... 28

Figure 8: Gel results of PERT assay on VLP isolates from 04/20/12...... 28

Figure 9: Gel results of PERT assay on 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 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

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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 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 by means of reverse transcriptase prior to transposition. DNA transposons, in contrast to their counterparts, do not use an RNA intermediate but a variety of different enzymes and mechanisms for transposition.

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Retrotransposons are further separated into two groups based on their sequence characteristics and are identified as LTR retrotransposons or non LTR retrotransposons, illustrated in figure 1 (Bannert 14575).

Figure 1: Transposable elements. A simplified schematic of the different classes of transposable elements (based on modified figure in Bannert, 2004).

LTR retrotransposons are flanked by identical long-terminal repeated sequences on their ends. Non-LTR retrotransposons include non-autonomous short interspersed elements

(SINES), that lack protein coding capability, and autonomous long interspersed elements

(LINES). LTR and non-LTR retrotransposons are further divided in different families based on sequence similarities (Bannert 14572). These retroelements, specifically LTR retrotransposons, are similar in sequence to retroviruses as they use the same mechanism of reverse transcription. Similar to HIV-1, open reading frames in LTR retrotransposons code for GAG, the structural capsid protein required in the formation of virus-like particles, and POL, which is comprised of protease, reverse transcriptase, integrase, and ! $!

ribonuclease H (Wicker 974). Given that SINES do not code for proteins, their sequences are truncated compared to their counterparts. In order to function, they must rely on the enzymes required for reverse transcription and transposition that are coded by

LINES (Bannert 14572) (Figure 2).

Figure 2: Structures of retroelements. A generalize graphic of the important sequence structures in SINE, LINE, and LTR retroelements. Arrows indicate repeated sequences

(Bannert, 2004).

Transposable element sequences are omnipresent in the genomes of all eukaryotic organisms and are credited with being the source of the vast variation in genome size, which is observable not only between the different taxa, but occasionally amongst closely related species as well (Feschotte 339). Up until the last couple of decades, transposable element sequences were often referred to as “junk DNA” or “selfish DNA.” As reviewed by Petrov (24), much of the conjecture was that the vast amount of non-coding “junk”

DNA present in genomes was simply inert, acting like a buffer-zone for genes, whilst ! %!

others speculated that these sequences were derived from the continuing accrual of transposable elements in a merely parasitic fashion. However, the present views have shifted into a more functional exploration of how transposable elements play a role in the shaping of genome evolution, how their activity may impart genetic diversity amongst species and populations, and how these elements may have a direct effect on gene expression. Instead of simply being referred to as “junk” or “selfish” DNA, transposable elements are now acknowledged as drivers of genome evolution (Biemont 1086,

Kazazian 1627).

Much of the DNA in the human genome and other genomes as well, is the result of transposable element activity, (Bannert 14572). There are obvious negative effects of having actively transposing elements present in host germ cells and predictable problems that can be caused by their insertional mutagenesis (Chiu 320). Human cells have many mechanisms in place to control transposition that are currently being explored. Inactivity of transposable elements can be caused by the buildup of inactivating mutations, epigenetic silencing through methylation, siRNA induced methylation or degradation of transcripts, and the activity of host cytidine deaminases, such as APOBEC3 (Bannert,

14578 Chiu, 343 Feschotte 340). Interestingly, it has even been suggested that, when compared to other mammals, the enhanced variability and expansion of the APOBEC3 gene family attained in primates over millions of years came as a result of the selective pressure to inhibit retrotransposon mobility (Chiu 343).

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A very popular focus in the world of transposable elements, as recently reviewed by Tenaillion (473), is centered on angiosperms, where the genome sizes can differ by as much as 1000-fold. Transposable elements, particularly those of retroelements, are highly predominant in angiosperms. In the review, Tenaillion points out that genome sizes of angiosperms follow a trend in which the quantity of transposable sequence content is positively correlated with the genome size (Figure 3) and observes that the content of these angiosperm genomes can range from less than 30% transposable element sequence, as seen in Arabidopsis thaliana, to almost 90% transposable elements in barley. The differential regulation of transposable elements and stress activation factors of transposition in plants play a major role in this phenomenon. In addition to epigenetic control, like siRNA mediated methylation, the unique mechanisms possessed by plants to manage transposition and the accumulation of transposable element sequences have been comprehensively reviewed (Kumar 483, Tenaillion 475, Vitte 17638). Plants are also capable of losing large amounts of transposable element sequences in genomic DNA via illegitimate recombination events on chromosomes and unequal intra-strand homologous recombination, in which whole sections of DNA are lost in-between flanking LTR sequences or in-between LTR sequences in different genomic locations (Vitte 17639).

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Figure 3: Diploid angiosperm genomes. The relative sizes of selected angiosperm genomes relative the level of transposable element sequence content (Tenaillion, 2010).

Members of the family Liliaceae are proving to be a very interesting target of study in the effort to further understand transposable element involvement in genome size variability and evolution. Species within Liliaceae show immense genome size variation, having the largest reported angiosperm genomes with a C value of up to130 pg, while some members have smaller genome sizes with C values closer to 4 pg, with a mean ! (!

species C value of 50 pg (Leitch 2230). For a practical comparison, the C value of the human genome is 3 pg. When referring to plant genome sizes, C values from 3.5 pg to

14 pg are regarded as intermediate, 14 pg to 35 pg are large, and genomes sizes greater than 35 pg are deemed “giant” genomes (Kelly 3). The massive genomes that are customary in the Liliaceae family are extreme examples of transposable element, specifically retroelement, proliferation. Species within this family provide an attractive model for study to further understanding of the mechanisms possessed by transposable elements to proliferate and evade host defenses, to elucidate the external stresses that initiate rapid transposable element proliferation events or reactivation of transposable elements, and to expose the evolutionary consequences of giant 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 extensively characterized. A recent study utilized shotgun sequencing methods and fosmid libraries that had been selected for highly repetitive sequences from two continentally separated Liliaceae species with similar genome size

(Ambrozova 258). Sequencing data identified various LTR retrotransposon sequences from a distinct lineage but they did not make up a predominant component of the repetitive DNA. Instead, the authors observed that the repetitive sequences originated from numerous diverse transposons families and that the large genome sizes of these two species must be due to a breakdown in the inherent control mechanisms and DNA removal methods in these subspecies. While other studies using next-generation sequencing methods are currently being carried out to characterize highly repetitive DNA ! )!

in (Kelly 4), there have not been published efforts to isolate and characterize active retroelements.

Transposable elements in the genome may be actively transposing or may be ancient remnants of once active families (Biemont 1087). Analyzing genome sequence data, such as Ambrozova (261), may elucidate what particular element types have historically been able to proliferate and to what extent. However, this sort of analysis does not confirm the presence actively transposing retroelements. The only means to establish that a retroelement is active is to isolate it in the form of virus-like particles.

Experiments to isolate and characterize active retroelements in the form of virus-like particles have been successfully carried out for grass species (Melcher 49, Muthukumar

169). This thesis research will use modified virus-like particle isolation techniques as described previously (Melcher 52). Virus-like particle nucleic acids will be purified for subsequent characterization using reverse transcription PCR and several primer sets that are specific to the reverse transcriptase gene of Gypsy-like and Copia-like LTR retroelements. This method was first tested using barley as a model system.

Barley is a member of the Triticeae, which includes other essential cereal crops like wheat and rye. Barley is the fourth most produced crop in the world, by weight, and ranks fourth in land usage (Zhou 3). Due to this fact, barley and its transposable elements have been highly characterized, and Triticeae are generally better characterized than any other plant genomes (Wicker 715). The sizeable amount of repetitive sequences in barley, which make up almost 90% of the genome (Tenaillion 473), has chiefly been attributed to a handful of transposable element families. Additionally, recent research has ! *!

shown that Copia BARE1 virus-like particles can be successfully isolated from barley homogenates using ultracentrifugation techniques (Jaaskelainen 3). For these reasons, and the relative ease with which barley can be obtained and grown, it was the model for carrying out the initial experimental aims of this research. Once successful results were achieved with barley, the same techniques were applied to isolate and attempt to characterize virus-like particles in species from the Family Liliaceae. The goal is to allow us to ascertain which retroelements are actively transposing in Liliaceae species and to what degree.

In an attempt to accomplish this goal, this thesis research has employed the specific experimental objectives listed in the next section.

Experimental Objectives

Specific Aim 1: To develop and test an ultracentrifugation technique to isolate virus-like particles from plant homogenates in such a way that conserves the activity of the retroelement reverse transcriptase enzymes.

Specific Aim 2: To use the Product-Enhanced Reverse Transcriptase assay to test for the presence of reverse transcriptase in the ultracentrifuged isolates which will be indicative of the presence of virus-like particles.

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Specific Aim 3: To isolate and quantify total RNA from the virus-like particle isolates.

Specific Aim 4: Characterize RNA purified from virus-like particle samples using reverse transcription PCR techniques.

Methods and Procedures

Specific Aim 1: Develop and test an ultracentrifugation technique to isolate virus- like particles

Preparation of barley homogenate: Barley cultivar Morex were obtained from Dr.

Timothy J. Close, from the Department of Botany and Plant Sciences at UC Riverside.

The seeds were grown under constant light for three to five weeks. Plant homogenate preparation and ultracentrifugation techniques were modified from an approach described by Melcher et al (49). For each trial, six individual samples were prepared by using barley leaf tips (100 mg to 150 mg). Barley leaf tips were added to a 1.5 ml standing screw cap tube containing (12) 2.5 mm sterile glass beads, 850 µl chilled 0.1M sodium citrate buffer (pH 7) and 80 µl 0.1M dithiothreitol. Tubes were left on ice for 10 minutes prior to homogenization. Tissues were disrupted at 5000 rpm in a mini bead beater in 30- second intervals for a total of two minutes. The tubes were kept on ice in between each disruption. The barley leaf homogenates were transferred to 6 individual sterile 1.5 ml ! ""!

microfuge tubes and centrifuged for no longer than 5 minutes at maximum speed (15,000 rpm) at room temperature on a tabletop centrifuge. The supernatants (800 µl) were immediately transferred to 6 new sterile 1.5 ml microfuge tubes and 80 µl of 33.3% (v/v)

Triton X-100 will be added. These tubes are briefly inverted and placed on ice.

Ultracentrifugation: The barley leaf homogenates (700 µl) were transferred to 1.5 ml polyallomer ultracentrifuge tubes (Beckman #357448) underlain with 700 µl chilled 20%

(w/v) sucrose solution in 0.1M sodium citrate buffer. Tubes were weighed individually before ultracentrifugation. A TLA-100.3 fixed angle rotor was fit with (6) 11 mm Delrin adapters (Beckman #355919). Using a Beckman TL-100 ultracentrifuge the tubes were subjected to ultracentrifugation at 36,000 rpm at 4°C for 45 minutes. Supernatants were carefully removed by aspiration and pellets are resuspended in 1400 µl chilled 0.5X sodium citrate buffer by pipetting. Tubes were centrifuged again at 12,000 rpm and 4°C for 10 minutes. A 1000 µl aliquot of the supernatants were removed from the top of the tube and added to new 1.5 ml polyallomer ultracentrifuge tubes underlain with 400 µl chilled 20% (w/v) sucrose solution in 0.1M sodium citrate buffer. Tubes were weighed and subjected to ultracentrifugation at 53,000 rpm and 4°C for 65 minutes. Supernatants were carefully removed by aspiration and VLP pellets are resuspended in 30 µl of chilled re-suspension buffer (50 mM KCl, 25 mM Tri-HCl pH 7.5, 5 mM DTT, 0.25 mM EDTA pH 8, 0.25% Triton X-100, 50% glycerol) (recipe Chang, 45).

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This same technique was applied to all fresh Liliales samples, collected fresh from specimens of different ages. Fresh leaf tips (100 mg – 150 mg) were frozen for later

RNA isolation from tissue.

Specific Aim 2: Use the Product-Enhanced Reverse Transcriptase assay to test for the presence of virus-like particles.

Verification of the presence of virus-like particles

The Product-Enhanced Reverse Transcriptase assay was used as described by

Chang (45).

A.) Primer/Template Reaction: In a primer/temple reaction mixture, 0.8 µg of

Bacteriophage MS2 genomic RNA (Roche, 0.8 µg/µl) is used as template and 1

µl of RT-1 primer [5’-d(CATAGGTCAAACCTCCTAGGAATG)-3’] (10 µM) is

used as primer. This is mixed with 2 µl of nuclease free water in a PCR tube.

Nine reaction mixtures are used. In one tube the MS2 Bacteriophage RNA is

omitted and the volume scaled up with nuclease free water to use as a negative

control. The tubes are placed in a thermocycler, heated at 85°C for 5 minutes,

annealed at 37°C for 30 minutes, and chilled on ice for five minutes prior to use.

B.) Reverse Transcription, first strand cDNA synthesis: The same primer/template

reactions tubes (containing 4 µl of primer/temple mix) are used for first strand ! "$!

cDNA synthesis. Added into each of the tubes is 1 µl dNTPs (10 µM each), 2 µl

0.1M DTT, 2 µl 10X RT buffer (Promega, 500 mM KCl, 100 mM Tris-HCl), and

4 µl MgCl2 (5 mM). Into six of the tubes is added 6 µl of resuspended VLP pellet

and 1 µl nuclease free water. As a negative control, one tube has an additional 7

µl of nuclease free water and lacks added reverse transcriptase. As a positive

control, one tube has 1 µl of Superscript III RT (Roche, 200 U/µl) as a source of

reverse transcriptase and 6 µl of nuclease free water. Another negative control is

made using the primer/template reaction tube in which the MS2 Bacteriophage

RNA was omitted. To this tube, 1 µl of Superscript III RT (Roche, 200 U/µl) and

6 µl of nuclease free water is added. Each tube will contain a total reaction

volume of 20 µl. These tubes will be placed in a thermocycler and incubated at

37°C for one hour. The cDNA can be used immediately as a template for PCR

amplification or frozen.

C.) PCR amplification and visualization of PCR product: In a 10 µl total reaction

volume, 1 µl of first strand cDNA is mixed with 5 µl of 2X PCR Master Mix

(Promega), 1 µl RNase A (Invitrogen, 12 µg/µl), 1 µl of RT-1 primer (10 µM), 1

µl of RT-2 primer [5’-d(TCCTGCTCAACTTCCTGTCGAG)-3’] (10 µM), and 1

µl of nuclease free water. The following PCR thermocycler parameters are used

for amplification:

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1. Denaturation - 94°C for 5 minutes

2. Denaturation - 94°C for 30 seconds

3. Primer annealing - 55° for 45 seconds 35 cycles

3. Extension - 72°C for 1 minute

4. Final extension - 72°C for 5 minutes

5. Hold - 4° forever

The 112bp PCR products are visualized alongside a low molecular weight

marker, such as NEB Low Molecular Weight Ladder or NEB 100bp Ladder,

on a 3.5% agarose gel run at 80 volts.

Specific Aim 3: Isolate and quantify total RNA from the virus-like particle isolates.

Isolation and quantification of RNA from virus-like particles and fresh leaf samples:

The total RNA was purified using a method that is suitable for cDNA synthesis and sequencing as a downstream application.

The RNeasy Plant Mini Kit (Qiagen #74903) was used to purify RNA virus-like particle samples and from frozen leaf tissue samples. This RNA purification technique does not prevent the copurification of contaminating plant genomic DNA fragments.

After the purification of RNA from the virus-like particle isolates and from leaf tissue ! "&!

samples, it was necessary to DNase treat the RNA samples. A commercial DNase, such as Ambion Turbo DNase (#AM2238) was used.

Complete inactivation of DNase activity in the treated RNA samples was confirmed by simple gel analysis. This was done by mixing 1 µl of treated RNA sample with 3 µl of any standard molecular weight marker and incubating the mixture at 37°C for 30 minutes. After incubation, 1 µl of gel loading dye was added to the sample and loaded on a 1% agarose gel along side the same untreated molecular weight marker. After ethidium bromide staining and visualization, remaining DNase activity in the treated samples is apparent by the smearing of the molecular weight marker.

Quantification of purified and DNase treated RNA was performed using a NanoDrop!

ND-3300 Fluorospectrometer. The manufacturer’s protocol was followed to perform a

RiboGreen! assay and create a standard curve for RNA in both low (5 – 50 ng/ml) and high (25 – 1000 ng/ml) quantitation ranges.

Specific Aim 4: Characterize purified RNA from virus-like particle samples using reverse transcription PCR techniques and sequencing by capillary electrophoresis.

cDNA synthesis from purified RNA: The DNase 1 treated RNA from virus-like particle samples and leaf samples was converted into cDNA pools using Invitrogen SuperScript

III Reverse Transcriptase Kit (# 18080-093) and following kit protocol for reactions primed by random hexamers. For all cDNA synthesis reactions, no reverse transcriptase controls were performed for every RNA sample. Additionally, a no-template control ! "'!

(substituting 1 µl molecular grade water for RNA) was run in parallel with every cDNA synthesis reaction carried out.

PCR reactions for retroelement reverse transcriptase using cDNA template: The cDNA pools were used as template for PCR reactions using several primers that are specific to the reverse transcriptase gene of Copia-like elements and Gypsy-like elements. As internal standards, primers for ubiquitin were used for barley samples and elongation factor " for Liliales samples. All of the PCR reactions were carried out as follows: in a

10 µl total reaction volume, 1 µl of first strand cDNA is combined with 5 µl of 2X PCR

Master Mix (Promega), 1 µl of forward primer (10 µM), 1 µl reverse primer (10 µM), and 2 µl of nuclease free water. PCR products were visualized on 3% agarose gels run at

100 constant volts.

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The following PCR thermocycler parameters were used for amplification:

Barley ubiquitin

Ubi Forward [5’-d(GCCGCACCCTCGCCGACTA)-3’]

Ubi Reverse [5’-d(CGGCGTTGGGGCACTCCTTC)-3’]

1. Denaturation - 94°C for 2 minutes

2. Denaturation - 94°C for 30 seconds

3. Primer annealing - 60° for 45 seconds 35 cycles

3. Extension - 72°C for 1 minute

4. Final extension - 72°C for 7 minutes

5. Hold - 4° forever

Lilium elongation factor "

Elf Forward [5’-d(GTCAAGGATCTGAAGCGTGG)-3’]

Elf Reverse [5’-d(GATCGCCTGTCAATCTTGGT)-3’]

1. Denaturation - 94°C for 2 minutes

2. Denaturation - 94°C for 30 seconds

3. Primer annealing - 55° for 45 seconds 35 cycles

3. Extension - 72°C for 1 minute

4. Final extension - 72°C for 7 minutes

5. Hold - 4° forever ! ")!

Copia-like reverse transcriptase gene

Copia Forward [5’-d(CARATGGARGTNAARAC)-3’]

Copia Reverse [5’-d(CATRTCRTCNACRTA)-3’]

1. Denaturation - 94°C for 2 minutes

2. Denaturation - 94°C for 1 minute

3. Primer annealing - 46° for 2 minutes 50 cycles (or 35 cycles)

3. Extension - 72°C for 3 minutes

4. Final extension - 72°C for 7 minutes

5. Hold - 4° forever

“Friesen” Gypsy-like reverse transcriptase gene

Friesen Forward [5’-d(MRNATGTGYGTNGAYTAYMG)-3’]

Friesen Reverse [5’-d(RCAYTTNSWNARYTTNGCR)-3’]

1. Denaturation - 94°C for 2 minutes

2. Denaturation - 94°C for 1 minute

3. Primer annealing - 53° for 1 minute 45 cycles

3. Extension - 72°C for 1 minute

4. Final extension - 72°C for 7 minutes

5. Hold - 4° forever ! "*!

“Old Gypsy” Gypsy-like reverse transcriptase gene

Gypsy Forward [5’-d(TAYCCIHTICCICGIATHGA)-3’]

Gypsy Reverse [5’-d(ARCATRTCRTCIACRTA)-3’]

1. Denaturation - 94°C for 7 minutes

2. Denaturation - 94°C for 1 minute

3. Primer annealing - 40° for 1 minute 35 cycles

3. Extension - 72°C for 3 minutes

4. Final extension - 72°C for 7 minutes

5. Hold - 4° forever

PCR clean-up for capillary gel electrophoresis sequencing: PCR products that are amplified successfully using Copia reverse transcriptase gene primers were prepared for subsequent sequencing by first carrying out a PCR product clean-up step. In individual

PCR tubes, 1 µl of ExoSAP-IT enzyme is added to 3 µl of PCR product. After being mixed by pipetting, the tubes are incubated in a thermocycler at 37°C for 30 minutes to allow removal of ssDNA and dNTPs by the ExoSAP-IT enzymes followed by a 15- minute incubation at 80°C to inactivate enzymes.

Dideoxy chain termination sequencing and clean-up: Chain termination sequencing was performed using Applied Biosystems Big Dye ! v3.1 kit. In the same ExoSAP-IT tubes, ! #+!

4 µl of Big Dye ! v3.1 reagent was added along with 2 µl of 10 µM of specific reverse primer. These tubes were placed in a thermocycler under the following conditions:

1. Denaturation - 96°C for 1 minute

2. Denaturation - 96°C for 10 seconds

3. Primer annealing - 53° for 5 seconds 25 cycles

3. Extension - 60°C for 4 minutes

4. Hold - 4° forever

For a final clean-up step of chain termination sequencing products, Applied Biosystems

XTerminator# kit and protocol were used. Following the chain termination reaction, wide-barrel pipette tips were used to add 10 µl of XTerminator# resin and 45 µl SAM solution into each of the tubes. The tubes were subsequently shaken in a MixMate mixer at 2000 rpm for 30 minutes to allow sequestration of unincorporated ddNTPs prior to sequencing. All samples were loaded according to manufacturer protocol on a 96 well plate for capillary gel electrophoresis on an Applied Biosystems 3130 Genetic Analyzer.

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Results and Discussion

Ultracentrifugation

This research utilized and modified existing virus-like particle isolation techniques for retroelement isolation. Plant leaf samples were homogenized in a manner designed in such a way to preserve the enzymatic activity of reverse transcriptase enzyme contained in the VLPs. These preparations were subjected to ultracentrifugation as previously described (Muthukumar 169, Lane 690). This is an ultracentrifugation method that is adequate to obtain clean VLP containing fractions given that specific criteria, such as sedimentation coefficient and sedimentation velocity, for the possible homogenous mix of VLPs was not known. Table 1 includes a list of all plant samples used for VLP isolation using this technique. These preparations were immediately sampled for the presence of active reverse transcriptase enzyme, which would be indicative of the successful isolation of enzyme containing VLPs.

Table 1: List of all fresh plant samples isolated by ultracentrifugation and the respective dates of the isolations. Note that barley was the principal plant sampled for these techniques.

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The Product Enhanced Reverse Transcriptase Assay

The Product Enhanced Reverse Transcriptase assay, or PERT, (Chang, 45) was employed in order to check the putative VLP containing ultracentrifugation fractions for the occurrence of active reverse transcriptase enzyme activity. The confirmation of the presence of reverse transcriptase activity in the collected ultracentrifugation samples would suggest the presence of active VLPs in the samples and in turn would confirm successful centrifugation protocol. In this protocol the first step is a primer/template reaction in which a DNA primer is allowed to complimentarily bind to MS2 phage RNA template and this will serve as the primed-template for the reverse transcription reaction

(cDNA synthesis). For the reverse transcription reaction it was arbitrarily chosen that 6

µl of resuspended VLP pellet would be used as a presumed source of reverse transcriptase. The final step is a simple PCR reaction of all samples using primers complimentary to a 112 base pair fragment of the newly synthesized MS2 phage cDNA and subsequent visualization on a 3.5% agarose gel. In this final step of the assay, the occurrence of the 112 base pair fragment in any PCR sample indicates that the original sample contained reverse transcriptase, the source being from a VLP sample or a commercial control enzyme. These methods were practiced in barley and a sample of the results is shown in figure 4 on the next page. The tubes that initially received 6 µl of ultracentrifuged VLP sample as a source of reverse transcriptase show more variable results; some PCR fragments are notably brighter than others while some appear to have very weak or negative results, as observed in lanes 3 through 8 in figure 4. In this gel it can also be observed that the addition of commercial reverse transcriptase as a positive ! #$!

control produces a very strong positive result with a very bright PCR fragment, as observed in gel lane 10 of figure 4.

Figure 4: Gel results of PERT assay performed on barley VLP isolates from

11/22/2011. PCR products observed on a 3.5% agarose gel.

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The variability in the VLP sample results may not necessarily be attributed to the simple presence or lack of active VLPs in the tested barley leaves as the samples were always collected from a single young plant. Rather, these results can perhaps be credited to slight variations in the manner in which each sample was handled during homogenation and ultracentrifugation.

This same technique was subsequently applied to all Liliales samples including

Lilium pardalinum, Prosartes smithii, , andrewsiana, Smilax californica, and Calochortus venustus. Initial samplings of Lilium pardalinum also display similar variations in the brightness of the PCR fragments that follow cDNA synthesis using VLP preparations as the source of reverse transcriptase, as observed in figures 2 and 3. In figure 5, faint product bands can be observed in lanes 5, 7 and 9, however, lanes 4 and 8 appear to be negative.

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Figure 5: Gel results of PERT assay performed on Lilium VLP isolates from

01/17/2012. PCR products observed on a 3.5% agarose gel.

! #'!

Likewise in figure 6, lanes 8, 10 and 11 all display product bands of varying brightness that were produced using VLP preparations, but lane 9, which also contained a VLP preparation, is negative.!!!

Figure 6: Gel results of PERT assay performed on Lilium VLP isolates from

01/31/2012. PCR products observed on a 3.5% agarose gel. Note the faint product band around 112 base pairs in lane 15. This is the first occurrence of possible reagent contamination leading to a false positive result. ! #(!

There were difficulties for a period of time, however, with false positive results. The first observed evidence of this is also seen in figure 6 where there is a faint product band visible in lane 15, which contained the no reverse transcriptase negative control for cDNA synthesis. The fragment size of this band is also approximately 112 base pairs.

This PCR fragment also occurs in no-template negative controls for PCR that omitted cDNA. The occurrence of this false positive result becomes even more pronounced in several succeeding reverse transcriptase assays using ultracentrifuged samples from

Scoliopus, Calochortus and Prosartes (see figure 7 lane 10, figure 8 lane 11, figure 9 lane

10). It is reasonable to venture that a reagent used in one of the three steps for the reverse transcriptase assay may have been, at some point, contaminated with the 112 base pair

PCR product. Additionally, in figures 4 and 5 for Scoliopus and Prosartes, all of the

PCR product bands for the VLP samples and the negative controls are of near equal brightness, which is highly indicative of PCR product contamination.

!

! #)!

Figure 7: Gel results of PERT assay Figure 8: Gel results of PERT

performed on Scoliopus VLP isolates assay performed on Prosartes

from 04/13/2012. PCR products VLP isolates from

observed on a 3.5% agarose gel. 04/20/2012. PCR products

False positive results caused by observed on a 3.5% agarose

presumed PCR product gel displayed the occurrence

contamination produced product of continued false positive

bands of equal intensities for all results for this assay.

samples, excluding the positive

control in lane 11, which is

significantly brighter.

! ! #*!

In figure 9, for Calochortus samples, many reagents were refreshed (see methods for reagents) and new RT-1 and RT-2 primer dilutions were made, albeit from the original stock, and the results once again display variation in the intensities of the PCR product bands from the VLP samples – lanes 9 and 10 for the no reverse transcriptase and no template controls, respectively, still show product bands of comparable intensity.

Figure 9: Gel results of PERT assay performed on Calochortus VLP isolates from

04/30/2012. PCR products observed on a 3.5% agarose gel. After refreshing most reagents used for cDNA synthesis and PCR, variation in intensity of sample PCR products is once again observed, but PCR products still persist in negative controls. ! $+!

At the period of time that these Liliales samples (listed previously) were being used for the PERT assay, over half a dozen assays had already been performed in the previous months using barley. Contamination by this 112 base pair PCR product would only need to be minimal, numbering just a few template molecules (McPherson 28), to confer false positive results. Figure 11 and 12 (discussed in greater detail later) will illustrate that this was the cause for these false positive results, the source being the primers (RT-1 and RT-

2) used for the reverse transcriptase assay protocol. These primers were later replaced with a new set and extra measures were taken to avoid contamination of the reagents by the small 112 base pair PCR product. The preventive measures included using a separate set of pipettes for preparing all reverse transcriptase assay mixtures and carrying out these preparation steps in a room separate from where the PCR reactions and gel electrophoresis would take place. Despite the incidence of false positive results for the reverse transcriptase assay performed on several all the plant species, Calochortus,

Scoliopus, Prosartes, and Smilax, their VLP containing ultracentrifuge fractions were still used for RNA isolation. New MS2 phage primer sets were ordered for the reverse transcriptase assay and these were shown to eliminate the occurrence of false positive results in no template and no reverse transcriptase control samples. Figure 10 shows a

3.5% agarose gel displaying the results of the reverse transcriptase assay on ultracentrifuged barley samples.

! $"!

Figure 10: Gel results of the PERT assay performed on barley VLP isolates from

12/10/2012. PCR products observed on a 3.5% agarose gel. The use of new RT-1 and

RT-2 primer stocks as well as other measure discussed to prevent reagent contamination, show absence of false positive PCR results and a successful PERT assay.

! $#!

All new PCR and cDNA synthesis reagents were used and the same measures to prevent template contamination were employed. Lane 9 in figure 10 corresponds to the no template control for cDNA synthesis and lane 11 corresponds to the no reverse transcriptase control for cDNA synthesis. It is clear that these lanes do not show PCR product at 112 base pairs. Additionally, the plant samples continued to show a slight variation in the brightness of the product bands (lane 4 being notably brighter) and the positive control containing commercial reverse transcriptase enzyme displays a significant amount of PCR product at 112 base pairs. Liliales samples have also been re- isolated and tested for reverse transcriptase activity by the graduate student continuing this project (data not shown). In addition to barley, her work has also confirmed the presence of reverse transcriptase activity for the same plant samples in which we had previously obtained ambiguous results due to this contamination problem.

RNA Purification and Quantification

After performing reverse transcriptase assays, the remainder of the resuspended

VLP pellets were conserved and stored at -80°C for later RNA isolation. Fresh leaf tips from the same Liliales samples collected during VLP isolation were also saved and stored at -80°C for RNA isolation. The stored RNA was purified and treated with DNase 1 enzyme. Following the DNase 1 treatments, RNA was quantified using both standard spectrophotometry, and RiboGreen fluorescent assays. Results from VLP samples and leaf tips are outlined in table 2. ! $$!

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;<>=F!!79;0!;G-;!.-4/0H!=-A>/0!&!89:;-<:0E!672!>99/0E!@49A!;I9!E<=;<:8;!.-4/0H!

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! All of the RNA concentrations were measured using a fluorospectrometer and were conducted at the same period of time using the same standards. The measurements in table 2 have been edited to exclude any outliers in the results from the fluorospectrometer. Of the resuspended VLP pellet samples, there were two with notably low RNA concentrations, including Prosartes and Scoliopus. It should be pointed out that the RNA concentrations acquired from fresh leaf samples for Prosartes and

Scoliopus were also quite low compared to other sampled plants, as seen in table 2.

These plants were each sampled twice for VLP isolation and not only were the RNA concentrations low compared to other samples, but the standard deviation between the concentration measurements was quite high. It is possible that large variation in the measurements between the two separate VLP preparations can be attributed to inaccuracies in the way each of the two separate samples were handled during the VLP isolation steps; however, the handling of the samples during fluorospectrometer measurements could be the contributing source of error between measurements for the same isolate. Additionally, it should also be noted that these types of errors would be very apparent when analyzing data with small sample sizes. RNA concentrations of

VLP preparations from barley, Lilium and Calochortus were reasonably consistent for all samples. The samples for Smilax and Clintonia are somewhere in between these two extremes, as noted in table 2.

! $&!

Reverse Transcription Reactions and PCR for Copia and Gypsy Reverse

Transcriptase Gene

The isolated RNA samples were used for reverse transcription PCR (RT-PCR) reactions. RT-PCR reactions were initially carried out on RNA from barley VLP and leaf preparations using both gene specific primers for the Gypsy-like reverse transcriptase genes and the Copia-like reverse transcriptase genes, and using random hexamers. The first experiment using barley samples was performed without using a negative control for

PCR. This is shown in figure 8. RT-PCR was performed on RNA from six barley VLP preparations from the same plant using gene-specific primers for the Gypsy-like reverse transcriptase gene (Friesen primers, see methods), but this yielded no positive PCR results. A no-template control for the reverse transcription reaction was done using

Gypsy Friesen primers and substituting molecular grade water for RNA and the positive control for the reverse transcription reaction using MS2 phage RNA as a template and

MS2 phage primers. There was a faint band observed in lane 12 of figure 11, which represents a 112 base pair fragment for the reverse transcriptase reaction positive control.

However, as a negative control for the PCR reaction had not been run in parallel and given the occurrence of false positive results in the previous reverse transcriptase assays, a separate gel was run with no template negative control for PCR using MS2 phage primers while omitting the addition of cDNA template. The same thermocycler conditions were followed for both PCR reactions (see methods). A bright band in lane 3 of the gel in figure 12 is observed. This 112 base pair fragment in lane 3 is observed despite the fact that this sample received no cDNA template. ! $'!

Figure 11: Gel results from 07/20/12 Figure 12: Gel results from 07/24/12 of a

of cDNA synthesis and PCR negative control PCR reaction performed

performed using Gypsy Friesen using RT-1 and RT-2 primers without

primers, RNA from barley VLP cDNA template. Lane 3 shows an

isolates and barley leaf samples. approximate 112 base pair PCR fragment.

Lane 12 shows positive cDNA PCR products observed on a 3.5% agarose

synthesis control using MS2 phage gel.

RNA, RT-1 and RT-2 primers. PCR !

products observed on a 3.5% agarose

gel.

! ! $(!

This result suggests that one of the PCR reagents used, most likely the MS2 phage primers, is contaminated with the 112 base pair PCR fragment. This would explicate the cause of the false positive results obtained for several of the reverse transcriptase assays, noted earlier. Additionally, these false positive results ceased in later reverse transcriptase assays that used new MS2 phage primer sets (refer back to figure 10).

As a control for successful DNase inactivation in the barley leaf RNA and VLP preparation RNA samples, a specific primer for barley ubiquitin was used for PCR after random primed cDNA synthesis. Figure 13 shows the gel results for ubiquitin PCR using barley cDNA samples as template. Lanes 2 and 3 contain barley VLP RNA as the source for cDNA template – neither of these lanes have visible bands at 219 base pairs, which is the expected fragment size for these ubiquitin primers. This is good evidence that the barley VLP preparations do not contain measurable amounts of contaminating cellular mRNA. Lane 4 corresponds to cDNA template from barley leaf RNA and, as expected, there was a bright band observed at approximately 219 base pairs. Lane 7 is the no reverse transcriptase control for the same RNA sample. There is a faint band visible at approximately 219 base pairs, which indicates that there may be some amount of genomic DNA that was not eliminated during DNase treatment of the sample. Following the results for this particular gel, all subsequent VLP preparation RNA and leaf RNA samples for all plants tested underwent extended DNase digestion. There was a very faint band observed in lane 5 – this lane corresponds to the no reverse transcriptase control from a barley VLP RNA sample. Although it could be speculated that there may ! $)!

be genomic DNA present in that sample, in this case it is likely that the very faint band is simply spillover from lane 4.

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. PCR products observed on a 3.5% agarose gel.

! $*!

! The previously discussed figure shows an example of these positive results using ubiquitin as a control for barley (figure 13). Figure 14 exhibits one example of elongation factor-" being used as a positive control for Liliales species.

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. PCR products observed on a 3% agarose gel. PCR products after cDNA synthesis are observed on Gel B. Products for all no reverse transcriptase controls and cDNA synthesis are observed on Gel A.

In figure 14 gel A, which contains the no reverse transcriptase controls for cDNA synthesis, it was observed that none of the lanes associated with VLP preparations ! %+!

displayed a 200 base pair fragment for elongation factor-". In the lanes corresponding to

Liliales leaf RNA samples, only lane 9 of gel A was observed to have a faint 200 base pair fragment. This no reverse transcriptase control is derived from DNase-treated total

Smilax leaf RNA. From these results it can be reasoned that there may be some small amount of genomic DNA remaining in this particular RNA sample. Gel B, in figure 14, is an agarose gel containing all of the products using Liliales VLP cDNA and leaf cDNA as template for elongation factor-" PCR. The brighter fragments observed in lanes 7 through 11 are derived from leaf RNA samples. Lanes 3 through 6 and lane 12 are derived from VLP RNA. These lanes represent Clintonia, Smilax, and Calochortus VLP

RNA and are free of visible PCR products; however, lanes 5 and 6 representing Prosartes and Scoliopus VLP RNA, do contain faint bands. While this may indicate some level of contamination of cellular RNAs in these specific preparations, the PCR product in lane 6 may be overflow from lane 7, which contains a very bright PCR band. It should also be noted that samples in lanes 7 through 9 of gel B in figure 14, which represent leaf RNA from Calochortus, Clintonia, and Smilax, have very bright PCR bands. Conversely, lanes

10 and 11 with Prosartes and Scoliopus leaf RNA samples, display more faint bands.

This coincides with the RNA concentration results shown in table 2 in which it is clear that Prosartes and Scoliopus leaf samples yielded lower RNA concentrations. The

Gypsy PCR products using the Friesen primers would have produced a band at around

410 base pairs and the PCR products using the “Old Gypsy” primers (see methods) would have produced a band at 260 base pairs; however, cDNA template derived from VLP

RNA samples and leaf RNA samples did not produce PCR product using either of the ! %"!

Gypsy primers following random-primed cDNA synthesis (data not shown). The negative results for the Gypsy PCR assays were obtained on more than one occasion.

Interestingly, these Gypsy primers have been used in Dr. Baysdorfer’s lab in the past for

PCR using DNA isolated from leaves of various Liliales species and have successfully produced PCR product. The results obtained from this project indicate that there were no

RNA sequences present in barley VLP, Liliales VLP, or leaf samples that were derived from the Gypsy reverse transcriptase gene that could have been detected using the

Friesen or “Old Gypsy” primers. This same assay was performed using primers specific to Copia reverse transcriptase for both barley and Liliales and the results were quite different. Figure 15 shows an example of the results for both Liliales VLP RNA and leaf

RNA samples. Despite the fact that the ubiquitin and elongation factor-" gels consistently show expected results for the no reverse transcriptase controls, which indicates the absence or near absence of contaminating genomic DNA (figures 13 and

14), the Copia gels do not show these same results. Figure 15, gel B is an agarose gel run for Copia PCR using Liliales random primed cDNA samples from VLP and leaf RNA.

All lanes display a significant amount of PCR fragment around 275 base pairs, which is expected for the Copia primers. When looking at the corresponding gel (figure 15, gel A) containing the no reverse transcriptase controls, all lanes also display a significant amount of PCR fragment around 275 base pairs. As the gels for the ubiquitin and elongation factor-" controls (figures 13 and 14) show successful DNase 1 digestion, the positive PCR results observed figure 15 require reasonable explanation. All RNA samples were treated with an extended DNase 1 digestion and the control gels (refer back ! %#!

to figure 14 with elongation factor-") show confirmation of successful degradation of contaminating genomic DNAs (excluding the Smilax leaf sample seen in lane 9 of gel A, figure 14).

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. PCR products observed on a 3% agarose gel. Gel B displays the PCR products after cDNA synthesis. Gel A displays the PCR products for the no reverse transcriptase controls.

! %$!

The PCR products observed for the no reverse transcriptase samples in figure 15, gel A, are consistent with what is expected using the Copia reverse transcriptase gene primers and there are two possibilities that may explain these results. It is possible that there is contamination, likely from cDNA, of the Copia reverse transcriptase sequence

(discussed further in sequencing results). Another possibility is the presence of the Copia reverse transcriptase sequence in the form of a DNA-RNA hybrid.

If there are active VLPs in the ultracentrifugation samples and in leaf RNA samples, it is possible that some of the nucleic acid contained in the VLPs exists in the form of a DNA-RNA hybrid. This should be expected in an active VLP as DNA-RNA hybrids are an intermediate of the reverse transcription reaction (Miller, 64). DNase 1 enzyme cannot fully degrade DNA in the form of a DNA-RNA hybrid and Invitrogen reports that DNase 1 activity on DNA-RNA hybrids is less than 1-2% of that for dsDNA

(Sutton, 473). Additionally, the Copia reverse transcriptase gene PCR parameters used for figure 15 required 50 amplification cycles, which makes it easy for even a small amount of contaminating DNA to be amplified. The PCR reaction was repeated changing the number of amplification cycles from 50 to 35. Another agarose gel of the

PCR products was run to see if these new PCR parameters would alter the previously observed results. The PCR products of the experimental samples (gel B) and the no reverse transcriptase controls (gel A) run at 35 amplification cycles are displayed in figure 16. Evidently, the product bands produced are much more faint at 35 PCR cycles compared to 50 PCR cycles (figure 15) and for one sample, the barley VLP control seen in lane 2 of figure 16 A, the product band has nearly disappeared. ! %%!

The faint multiple bands of non-specific products are no longer visible in figure 16 although they were previously observed in some of the sample lanes of both gels in figure

15. All of the samples investigated, either derived from VLP RNA or total leaf RNA, have the potential to be contaminated by very small amounts of genomic DNA, even after

DNase 1 treatment. To characterize the content of these PCR products, they were sequenced on an Applied Biosystems 3130 Genetic Analyzer capillary electrophoresis system.

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. PCR products observed on a 3% agarose gel.

Gel B displays PCR products after cDNA synthesis. Gel A displays the PCR products for the no reverse transcriptase controls. ! %&!

Sequencing and Analysis of Copia Reverse Transcriptase PCR Products

Sequencing of all of the PCR products in figures 15 and 16, gels A and B, was performed as described (see methods). The initial sequencing of Copia reverse transcriptase gene PCR products derived after 50 cycle amplifications (figure 15) yielded highly mixed sequence results (data not shown). Subsequently all PCR products that underwent 35 amplification cycles (from figure 16) were sequenced in hopes of acquiring cleaner data. These samples also yielded mixed sequences with the levels of heterogeneity varying between individual samples. One example of the sequencing traces is shown in figure 17. This figure displays a reasonably clean portion of the results for Smilax VLP, one of the best looking sequences we were able to obtain.

Figure 17: A sample of the sequence obtained from Copia RT-PCR products using

Smilax VLP RNA, displayed using 4Peaks software. This portion of sequence represents one of the best sequencing results obtained. Although the traces are strong, they are fairly heterogeneous, displaying at least two sequences.

! %'!

Although the sequence data was mixed, approximately half of the samples could still be analyzed to some extent with a fair degree of confidence when using the best portions of the sequence. Using NCBI Basic Local Alignment Search Tool, specifically using tblastx to search a translated nucleotide query against a translated nucleotide database, all samples were evaluated for the types of hits that could be found. A variety of hits were obtained using the database but all were comparable to either Copia-like reverse transcriptase sequences from various cereal genomic DNA or cereal-derived genomic clones – all from the Poaceae family. As there currently is an absence of sequence data representative of Liliaceae samples in the NCBI database, it was not expected to obtain hits from Liliaceae during this analysis; however, many of the sample sequences, including both VLP and leaf tissue samples, appeared to have a maximum similarity to genomic Copia-like reverse transcriptase sequences from barley when searched against the NCBI database. Several of the no reverse transcriptase control samples (Scoliopus VLP, Clintonia VLP, Calochortus leaf) that had a clean enough sequence to analyze displayed similarity to barley genomic Copia-like reverse transcriptase sequences. In addition to using NCBI Blast, we were also able to make use of some newly acquired genomic sequence data as Dr. Baysdorfer’s lab has recently been utilizing next generation sequencing techniques to acquire genome sequence data for

Liliaceae species. Using Sequencher software, we were able to use some of the genomic sequence data acquired for Smilax to attempt to align our VLP derived samples (data not shown). Unfortunately we were not successful aligning any of the Liliaceae samples.

Due to these results we speculated that, by some means, the reagents used to produce the ! %(!

sequenced samples had been contaminated with barley Copia reverse transcriptase sequence. As the obtained sequences were all quite mixed, the barley Copia reverse transcriptase sequence does not, by any means, represent all of the sequence data.

However, it does appear that in approximately half of the samples, it is the greatest source of the sequence signals and causes the sequencing results to be ambiguous. These results were obtained several times using different cDNA samples leading us to believe that our single Copia reverse transcriptase primer stock may have been contaminated with barley cDNA.

Our original hypothesis, prior to sequencing, to explain the presence of PCR products for the no reverse transcriptase controls observed in figures 15 and 16, was the presence of DNA-RNA hybrids within the VLPs. The occurrence of DNA-RNA hybrids could be expected in an active VLP as an intermediary of reverse transcription reactions

(Miller 64) and the hybrid would be unsusceptible to DNase1 enzyme digestion.

Subsequent to sequencing, however, it is apparent that the greatest source of the sequence signals are quite similar to barley Copia reverse transcriptase gene sequence which is likely due to the contamination of reagents by barley cDNA. Copia-primed and random- primed cDNA synthesis had been performed on barley VLP isolate RNA and barley leaf

RNA multiple times over the course of this project. How this contamination occurred is unknown, and unfortunately, this was not our first problem with the contamination of reagents. This hypothesis would, nevertheless, be a reasonable explanation for the source of the PCR products obtained in figure 16 A and 15 A for the no reverse transcriptase negative controls. If these reagents had been contaminated with barley cDNA, the Copia ! %)!

reverse transcriptase gene sequence could have come from several sources. The origin of the sequence may have been the genomic RNA in the barley VLP isolates if, in fact, those isolates contained active Copia elements, which are the most prevalent elements in the barley genome. It is also possible that the contamination may have originated from the barley leaf RNA that would contain reverse transcriptase transcripts for BARE1

(Jaaskelainen 2), the predominant autonomous Copia element in barley. In addition, if any barley VLP or leaf RNA samples contained very small amounts of undigested genomic DNA, this could also be a source of Copia reverse transcriptase sequence as almost 10% of the barley genome is made up of copies of the BARE Copia family

(Wicker 518).

! %*!

Conclusions and Future Research

Transposable elements are ubiquitous among the genomes of all eukaryotic organisms. LTR retrotransposons, in particular, are the major component of plant genomes (Wicker 518) and are credited as the source of the great genome size variation among angiosperms. The angiosperm family, Liliaceae, in the order Liliales, is observed as having some the largest genomes among angiosperms, often referred to as having

“giant” genomes. Among different Liliaceae species, genome sizes can differ significantly with a minimum 1C value of roughly 2 pg up to a 1C value as high 130 pg

(Leitch 6). This attribute makes Liliaceae an ideal focus of study to understand the effects of uncontrolled transposable element proliferation and to learn what factors lead to uncontrolled proliferation.

This thesis study utilized barley as a model to develop and test a virus-like particle isolation technique using ultracentrifugation and utilizing the Product Enhanced

Reverse Transcriptase (PERT) assay as a confirmation for successful isolation. If virus- like particle isolations techniques proved successful, the aim was to employ these techniques to sample and isolate active LTR retrotransposons in the form of virus-like particle from specific members of Liliaceae the Liliales family. The isolation techniques used in this thesis study have been successful for barley and Liliales samples. Positive

PERT assay results preceded RNA isolation and quantification from virus-like particle isolates. The long-term goal is to characterize the isolated virus-like particle RNA in order to elucidate precisely which LTR retrotransposons are active in each sampled ! &+!

Liliales species. Our reverse transcription PCR methods using specific primers for retroelement reverse transcriptase genes yielded ambiguous results.

Rather than continue using reverse transcription PCR and specific primers, which would require time-consuming optimization for each primer set used, we plan to examine the use of next generation sequencing as a more high throughput method for characterization. Our lab possesses the Ion Torrent Personal Genome Machine, a next generation sequencer that uses semiconductor chip technology. The Ion Torrent PGM possesses technology that can be used to get 10-1,000 MB of sequence per each 2 hour run (Delseny 410). The next goal is to use our barley virus-like particle RNA isolates to determine which preparation technique will yield the best sequencing results from the Ion

Torrent and then to apply the same methods to characterize our Liliaceae virus-like particle RNA isolates. Successful characterization will allow us to ascertain which LTR retroelements are actively transposing in each individual Liliales species and to what degree.

! &"!

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