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Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco

by
Justin Kirke

A Thesis Submitted to the Faculty of
The Charles E. Schmidt College of Science
In Partial Fulfillment of the Requirements for the Degree of
Master of Science

Florida Atlantic University
Boca Raton, FL December 2019
Copyright 2019 by Justin Kirke ii

Abstract

Author: Title:
Justin Kirke Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco

Institution: Thesis Advisor: Degree:
Florida Atlantic University Dr. Xing-Hai Zhang Master of Science

  • 2019
  • Year:

DNA damage is one of the most harmful stress inducers in living organisms.
Studies have shown that exposure to high doses of various types of radiation cause DNA sequence changes (mutation) and disturb protein synthesis, hormone balance, leaf gas exchange and enzyme activity. Recent discovery of a protein called Damage Suppressor Protein (Dsup), found in the tardigrade species Ramazzotius varieornatus, has shown to reduce the effects of radiation damage in human cell lines. We have generated multiple lines of tobacco plants expressing the Dsup gene and preformed numerous tests to show viability and response of these transgenic plants when exposed to mutagenic chemicals, UV radiation and ionizing radiation. We have also investigated Dsup function in association to DNA damage and repair in plants by analyzing the expression of related genes using RT-qPCR. We have also analyzed DNA damage from X-ray and UV treatments using an Alkaline Comet Assay. This project has the potential to help generate plants that are tolerant to more extreme stress environments, particularly DNA damage and

iv mutation, unshielded by our atmosphere. The possibility of growing plants accompanying human space travel and extraterrestrial colonization inspires our imagination. Extremotolerant tardigrade genes such as Dsup may be a valuable avenue in helping to cultivate crops in these future endeavors.

v

Acknowledgements

I would like to start by acknowledging my thesis advisor and mentor Dr. Xing-Hai
Zhang. Without his insight and guidance, I would not be where I am today. I would also like to thank my other committee members Dr. Mary Jane Sunders and Dr. David Binninger for their additional guidance and thoughtful input into my project.
I would also like to thank my coworkers and students who volunteered to help during the completion of this work. My fellow lab mates, Paveena Vichyavichien, and Xiao-Lu Jin. My direct independent research students, Tahoe Albergo, Andrew Balsamo, Nicholas Nifakos, Milove Jeannot, Amanda Lam, Ronscardy Mondesir, Andrew Adeyiga, Mohamed Abutineh, and Nicholas Pizzo.
Next, I would like to thank the Department of Biology, the college of medicine, and the FAU high school with all of their help to complete this achievement, including technical support and allowing me the use of equipment require to conduct my study.
I would like to thank my friends and family. Without the emotional support and encouragement of my parents and my life partner Jennifer Castignoli, I would not be where I am at today.
Lastly, I would also like to thank my scholarly Dungeons and Dragons crew, Amy
Makler, Josh Disatham, Douglas Holmes, Gurtejpal Ghuman, Sean Paz, Anthony Muscarella, and Erick España for helping distract me from the stress of this journey with another one.

vi

Analysis of Tardigrade Damage Suppressor Protein (Dsup) Expressed in Tobacco

List of Tables ..................................................................................................................... ix List of Figures..................................................................................................................... x Introduction......................................................................................................................... 1
DNA Damage and Repair in plants ................................................................................ 1

Damage Suppressor Protein (Dsup) from Tardigrade .................................................... 3 Single Cell Gel Electrophoresis (Alkaline Comet Assay) .............................................. 4 Dsup expression in plants ............................................................................................... 5
Methods and Materials........................................................................................................ 6
Generation of Transgenic Plants..................................................................................... 6

Growth Analysis ............................................................................................................. 8 Histochemical Staining analysis ..................................................................................... 8 DNA and RNA extraction, cDNA synthesis, and qPCR analysis .................................. 8 Analysis of DNA Damage Targets through RT-qPCR (Damage Assay)....................... 9 Single Cell Gel Electrophoresis (Alkaline Comet Assay) ............................................ 10
Results............................................................................................................................... 12
Bioinformatics Analysis................................................................................................ 12

Genotyping and Expression of Dsup Transgenic Plants............................................... 14 Histochemical Staining analysis ................................................................................... 15 vii
Growth Assay................................................................................................................ 16 Analysis of DNA Damage Targets by RT-qPCR (Damage Assay) ............................. 19 Single Cell Gel Electrophoresis (Alkaline Comet Assay) ............................................ 27
Discussion......................................................................................................................... 31 References......................................................................................................................... 36

viii

List of Tables

Table 1: Target genes tested for expression level changes in Dsup vs Control plants

treated with genomutagens. ↓ showed downregulation, ↑ showed upregulation,

and ≌ showed no change in gene expression. ............................................................ 20 ix

List of Figures

Figure 1: Engineered Ti plasmid containing Dsup and NPTII genes driven by NOS and CaMV-35S promoters............................................................................................ 7

Figure 2: Amino acid sequence for Dsup protein based off ascension “P0DOW4”.

Highlighted in yellow is the N-terminus, underlined is the α-helical region, and highlighted in green is the C-terminus. Boxed in Red is a possible nuclear localization signal (NLS) ............................................................................................ 12
Figure 3:I-TASSER Predicted 3D structure of the Dsup Protein ..................................... 13 Figure 4: pBlast results for α-helical region of the Dsup protein ..................................... 13 Figure 5: Relative gene dosage of Dsup gene for T0 plants based on reference gene
EFα.............................................................................................................................. 14
Figure 6: Relative expression of Dsup mRNA in T0 plants based on reference gene
EFα.............................................................................................................................. 14
Figure 7: Relative gene dosage of Dsup gene for T1 plants based on reference gene
EFα.............................................................................................................................. 15
Figure 8: GUS stain analysis of T0 tissue regeneration on EMS media........................... 16 Figure 9: Average fresh weight of EMS treated samples. *Significant difference found between treatment samples with p-value < 0.05. p-value = 0.0001262 ........... 17
Figure 10: Average root length of EMS treated samples. *Significant difference found between treatment samples with p-value > 0.05. p-value = 7.465e-05. ........... 18

x
Figure 11: Growth assay plates for RM1 under 0 mM, 1 mM and 3 mM EMS treatment. .................................................................................................................... 19
Figure 12: Growth assay plates for R5-3 under 0 mM, 1 mM and 3 mM EMS treatment. .................................................................................................................... 19
Figure 13: Relative gene expression of ATR. Example of upregulation between treated samples............................................................................................................ 21
Figure 14: Relative gene expression of TGA. Example of downregulation between treated samples............................................................................................................ 21
Figure 15:Relative gene expression of CASP5/6. Example of downregulation to normal levels between treated samples....................................................................... 22
Figure 16: Relative gene expression of Ku70. Example of no change in regulation between treated samples. ............................................................................................ 22
Figure 17: Relative gene expression of ERCC. Example of downregulation to normal levels between treated samples................................................................................... 23
Figure 18: Relative gene expression of TGA. Example of downregulation to normal levels between treated samples................................................................................... 23
Figure 19: Relative gene expression of RAD51. Example of upregulation between treated samples............................................................................................................ 24
Figure 20: Relative gene expression of DDB. Example of no change in regulation between treated samples. ............................................................................................ 24
Figure 21: Relative gene expression of ATM. Example of downregulation to normal levels between treated samples................................................................................... 25

xi
Figure 22: Relative gene expression of ATR. Example of upregulation between treated samples............................................................................................................ 25
Figure 23: Relative gene expression of TGA. Example of upregulation between treated samples............................................................................................................ 26
Figure 24: Relative gene expression of CEN2. Example of no change in regulation between treated samples. ............................................................................................ 26
Figure 25: Alkaline comet assay images for RM1 samples.............................................. 27 Figure 26: Alkaline comet assay images for R5-3 samples.............................................. 28 Figure 27: Average tail movement for UV-C treated samples. *Significant difference found between treatment samples with p-value < 0.05. p-value = 2.2e-16 ................ 28
Figure 28: Average tail movement for X-ray treated samples. *Significant difference found between treatment samples with p-value < 0.05. p-value = 9.221e-09 ............ 29

xii

Introduction

DNA Damage and Repair in plants
Mechanisms to repair DNA damage are important to the health, growth and development of any plant. While plants have many mechanisms to protect themselves from damage to the genome, it is still possible to cause significant changes in protein synthesis and disrupt natural development [1]. Most natural genome damage in plants can be attributed to two common sources. The sun, in the form of ultraviolet (UV) radiation, and from oxygen, creating reactive oxygen species (ROS) [2][3]. Additionally, anthropogenic or naturally occurring ionizing radiation and alkylation damage from mutagenic chemicals

  • play an important role in creating DNA mutations [4][5]
  • .

UV-induced damage is a common issue when considering damage to epidermal tissue in plants and animals. The most common forms from the sun that can reach the earth surface, UV-A (wavelength 320 – 400 nm) and UV-B (280 – 320 nm), can penetrate our atmosphere. The more intense UV-C (< 280 nm) is unable to penetrate and is less of a threat to epidermal cells in an organism. UV-induced DNA damage creates pyrimidine dimers which lead to the halt of DNA replication and transcription. The dimers can hinder the progress of DNA polymerase and RNA polymerase II, causing a complete halt in transcription of affected genes. Since sunlight containing UV-A and UV-B is required for the development of plants, they have adapted by creating flavonoid pigments that effectively absorb this UV radiation. In plants this damage can also be reversed by means of direct DNA damage repair known as photoreactivation. The photolyase enzyme directly
1attaches to the dimer through activation by photons in the wavelength range of 320 – 450 nm, thereby removing the dimers and reversing the damage [4].
Ionizing radiation (IR) can be the cause of serval different DNA lesions. The damage can be contributed to lack of a specific target. The most common reaction associated in IR is its interaction with water. It can destabilize H2O molecules creating hydroxyl radicals, one of the ROS mentioned earlier. These hydroxyl radicals can attack the phosphate backbone of DNA and cause single stranded breaks [4]. This gives the hydroxyl radicals an opportunity to oxidize specific bases since they are no longer shielded by the double helix conformation. The most significant base to be affected is guanine, creating 8-hydroxyguanine, which can easily pair with both adenine and cytosine bases and can be recognized by varying polymerases for replication and transcription [4]. Singlestranded breaks (SSB) are repaired by means of DNA ligase. IR and UV-C can also lead to double- stranded breaks (DSB), which are more difficult to repair due to random ligation

of either end by DNA ligases, causing possible “illegitimate recombination”, resulting in

nucleotide sequence alterations (mutation) [4].
Alkylation damage is the spontaneous methylation of bases that can cause mismatched pairing. Most cells have extensive pathways to manage DNA damage with the most common cause being endogenous methylations agents [4][5]. The nuclear excision repair (NER), base excision repair (BER), and mismatch repair (MMR) pathways are some of the key mechanisms for repairing alkylation damage [6][7]. The alkylating agent, ethylmethane sulfonate (EMS), is commonly used in genetics research to induce point mutations in plants [8]. Most pairing caused by EMS mutation will either not change pairing preference, but some do the exact opposite and will not allow pairing with any base, usually

2leading to a halt in replication, transcription and translation. On occasion it will create the product, O6-alkylguanine, which will bind freely to adenine. After a successful replication,

  • this will cause a guanine to adenine point mutation [4] [8]
  • .

In the present study, the genomutagenic compound bleomycin (BLM) was used to induce DNA damage. This compound is used as a chemotherapy drug to battle lymphomas, neck, head and testicular cancers [9]. When bleomycin interacts with O2 and a metal (usually Fe(II) or Cu(I)) it is converted into its active form, active bleomycin (ABLM). ABLM can bind to DNA and cleave G-C bonds, causing either single or double stranded breaks. This compound is a good substitute to simulate ionizing radiation due to the similar symptoms

  • and pathways for DNA repair [10][11]
  • .

Damage Suppressor Protein (Dsup) from Tardigrade
The tardigrade, also known as water bear, are considered by many to be the world’s most hardy animal. Measuring in at 1 mm in length at their largest, these tiny creatures can withstand a plethora of extreme environments and are found in almost all aquatic environments all over the earth. When they are introduced to a dry environment, they enter an anhydrobiotic state. In a desiccated state, they can withstand extreme temperatures, potentially toxic chemicals, ionizing radiation, UV radiation and have even been known to survive in the vacuum of space [12]. This animal is now being used as a model organism to study the rigorous effects of space travel due to the complex nature of this microscopic

  • organism [13]
  • .

This organism’s ability to defend itself from these extreme environments caught

the attention of a research group from Japan. A team led by Takekazu Kunieda isolated unique chromatin-associated proteins found in an extremotolerant species of tardigrade

3called Ramazzotius varieornatus. They expressed these proteins in Drosophila cell cultures using a GFP tag and discovered a single protein called Damage Suppressor Protein (Dsup) which showed nuclear localization. The Dsup gene was introduced and expressed in the HEK293 human cell lines [14][15]. Transformed and untransformed cell lines were X-ray irradiated followed by analysis of DNA damage using an alkaline comet assay. The assay places cells in an alkaline solution to denature double-stranded DNA followed by single cell electrophoresis to measure single stranded breaks in the nuclear genome. The longer

the “tail” the more damage done to the DNA. Tails found in Dsup expressing cells were

  • reduced by 50% compared to the untransformed cells [14][15]
  • .

Samples were then irradiated and subjected to a γ-H2AX immunofluorescent assay.

When double stranded breaks occur, H2AX histones become phosphorylated and can be visualized by immunofluorescence. Fluorescent foci were counted and the Dsup expressing cell lines showed a 40% reduction in observed foci. Under treatments of high X-ray radiation, cells were also tested for viability. Untransformed cells showed abnormal round morphology while the Dsup expressing cells showed normal morphology. Dsup cells also showed a slightly faster proliferation rate compared to untransformed cell lines. This evidence shows that Dsup transformed cell lines had a greater viability compared to their untransformed counter parts when irradiated under X-ray [14][15]. Single Cell Gel Electrophoresis (Alkaline Comet Assay)

  • The alkaline comet assay was first published in 1984 by Ostling and Johanson [16]
  • .

It has become a widely used technology to access damage by single stranded and double stranded breaks to nuclear genomes in eukaryotic cells [17]. New protocols have even

4studied NER and BER repair pathways by allowing cells to repair before cell lysis is

  • conducted [18]
  • .

Briefly, isolated single cells are placed into agarose gel and lysed. This solidifies nuclei in the gel where the intact cell once previously. Samples are then washed and subjected to gel electrophoresis to separate smaller fragments of DNA from the larger undamaged sections of the genome. The gel is then dyed with a fluorescent DNA binding chemical and viewed under a fluorescent microscope. This shows a visible signal as a

“head” where the nucleus is located and a “tail” of damaged DNA running from the head.

The longer the tail the more breaks that are present in the nucleus. For our experiments we have modified multiple protocols in order to create a lysis/electrophoresis buffer

  • combination most suited to our needs working with tobacco [19][20]
  • .

Dsup expression in plants
Based on the observation of the effects of Dsup on human cell lines, we wondered what effect the tardigrade Dsup protein might have on plants growth and resistance to DNA mutation and other stresses, as well as its effect on expression of the endogenous, DNA damage response and repair related genes. We hypothesize that the presence of the Dsup protein may lead to reduction of DNA damage caused by genomutagenic sources in plants expressing this protein. We have used Agrobacterium mediated transformation method to create tobacco plants that express Dsup. This study was conducted by means of molecular and plant growth analysis. We have also developed our own optimized protocol for an alkaline comet assay to assess the viability of our Dsup lines in comparison to control lines.

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    University of Richmond UR Scholarship Repository Biology Faculty Publications Biology 1-25-2008 What Do Students Have to Say About Ecology and Evolution? Using Podcasting to Apply Integrative Biology Themes Across the Tree of Life Amy M. Treonis University of Richmond, [email protected] Malcolm Hill University of Richmond, [email protected] Theresa Dolson University of Richmond, [email protected] Sue McGinnis Elizabeth Miles Follow this and additional works at: http://scholarship.richmond.edu/biology-faculty-publications Part of the Biodiversity Commons, Biology Commons, and the Curriculum and Instruction Commons Recommended Citation Treonis, Amy, Malcolm Hill, Theresa Dolson, Sue McGinnis, and Elizabeth Miles. "What Do Students Have to Say About Ecology and Evolution? Using Podcasting to Apply Integrative Biology Themes Across the Tree of Life." MicrobeLibrary, January 25, 2008, 1-32. This Article is brought to you for free and open access by the Biology at UR Scholarship Repository. It has been accepted for inclusion in Biology Faculty Publications by an authorized administrator of UR Scholarship Repository. For more information, please contact [email protected]. MicrobeLibrary http://archive.microbelibrary.org/edzine/details_print.asp?id=2758&lang= What Do Students Have to Say About Ecology and Evolution? Using Podcasting to Apply Integrative Biology Themes Across the Tree of Life Resource Type: Curriculum: Classroom Publication Date: 1/25/2008 Authors Amy Treonis Department of Biology University of Richmond
  • Arthrotardigrada, Styraconyxidae) with Unique Pockets on the Legs

    Arthrotardigrada, Styraconyxidae) with Unique Pockets on the Legs

    Zoosyst. Evol. 96 (1) 2020, 115–122 | DOI 10.3897/zse.96.49676 A new marine tardigrade genus and species (Arthrotardigrada, Styraconyxidae) with unique pockets on the legs Shinta Fujimoto1, Naoto Jimi2 1 Research Center for Marine Biology, Graduate School of Life Sciences, Tohoku University, 9 Sakamoto, Aomori, Aomori 039-3501, Japan 2 National Institute of Polar Research, 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan http://zoobank.org/44F42F00-8B76-4C95-961D-4FC588C50BF0 Corresponding author: Shinta Fujimoto ([email protected]) Academic editor: Pavel Stoev ♦ Received 26 December 2019 ♦ Accepted 25 February 2020 ♦ Published 23 March 2020 Abstract A marine heterotardigrade Cyaegharctus kitamurai gen. et sp. nov. (Arthrotardigrada, Styraconyxidae) is described from Daidokut- su, a submarine cave off Iejima island, Okinawa Islands, Ryukyu Archipelago, Japan. It is easily distinguished from all other styr- aconyxids by its pocket organs (putative sensory structures) on all legs in addition to the usual leg sensory organs. Its combination of other character states, such as the dorso-ventrally flattened body, ovoid primary clavae, conical secondary clavae, large terminal anus, internal digits with proximal pads and peduncles, external digits with developed peduncles and all digits with three-pointed claws in adult female, supports the erection of a new genus and species. Key Words meiofauna, Pacific Ocean, sensory organs, submarine cave, Tardigrada Introduction et al. 2020). In addition to these ten genera, Fujimoto et al. (2017) reported an undescribed genus related to Styr- Marine tardigrades, specifically arthrotardigrades, exhib- aconyx and Tetrakentron from a submarine cave in Japan it remarkable morphological diversity (see comprehen- (for detail of this cave see Yamamoto et al.
  • Articles Is Proposed to Explain Their Domi- Cyanobacteria and Eukaryotic Microalgae (Microalgae) and Nance in Cryoconites Further Away from the Glacier Margins

    Articles Is Proposed to Explain Their Domi- Cyanobacteria and Eukaryotic Microalgae (Microalgae) and Nance in Cryoconites Further Away from the Glacier Margins

    Biogeosciences, 13, 659–674, 2016 www.biogeosciences.net/13/659/2016/ doi:10.5194/bg-13-659-2016 © Author(s) 2016. CC Attribution 3.0 License. Controls on microalgal community structures in cryoconite holes upon high-Arctic glaciers, Svalbard T. R. Vonnahme1,2,a, M. Devetter1,3, J. D. Žárský1,4, M. Šabacká1,5, and J. Elster1,6 1Centre for Polar Ecology, Faculty of Science, University of South Bohemia, Ceskéˇ Budejovice,ˇ Czech Republic 2University of Konstanz, Constance, Germany 3Biology Centre of the Academy of Science of the Czech Republic, Institute of Soil Biology, Ceskéˇ Budejovice,ˇ Czech Republic 4Department of Ecology, Charles University, Prague, Czech Republic 5British Antarctic Survey, Cambridge, UK 6Institute of Botany, Academy of the Science of the Czech Republic, Treboˇ n,ˇ Czech Republic anow at: Max Planck Institute for Marine Microbiology, Bremen, Germany Correspondence to: J. Elster ([email protected]) Received: 27 May 2015 – Published in Biogeosciences Discuss.: 29 July 2015 Revised: 8 January 2016 – Accepted: 14 January 2016 – Published: 3 February 2016 Abstract. Glaciers are known to harbor surprisingly com- with high sediment loads, high N : P ratios, and a high impact plex ecosystems. On their surface, distinct cylindrical holes of nutrient input by bird guano, as a proxy for nutrients. In filled with meltwater and sediments are considered hot spots these environments autochthonous nitrogen fixation appears for microbial life. The present paper addresses possible bi- to be negligible. Selective wind transport of Oscillatoriales ological interactions within the community of prokaryotic via soil and dust particles is proposed to explain their domi- cyanobacteria and eukaryotic microalgae (microalgae) and nance in cryoconites further away from the glacier margins.