________________________________________________________________________ Exploring the rns gene landscape in ophiostomatoid fungi and related taxa: Molecular characterization of mobile genetic elements and biochemical characterization of intron-encoded homing endonucleases. By Mohamed Hafez Ahmed Abdel-Fattah A Thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfilment of the requirements of the degree of: DOCTOR OF PHILOSOPHY Department of Microbiology Faculty of Science University of Manitoba Winnipeg, Manitoba Canada Copyright © 2012 by Mohamed Hafez Ahmed Abdel-Fattah ________________________________________________________________________ ABSTRACT The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon- optimized versions of I-OmiI and I-OmiII sequences were synthesized based on II ________________________________________________________________________ differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs. III ________________________________________________________________________ ACKNOWLEDGEMENTS I would like to express my gratitude to all those who gave me the possibility to complete this thesis specially my advisor Dr. Georg Hausner for giving me the opportunity to work in his lab and supervising my PhD project. This thesis would not have been possible without his support, guidance and encouragement. I would like to thank my thesis supervision committee members, Dr. Deborah Court, Dr. Ivan Oresnik and Dr. Michele Piercey-Normore for their help, support, encouragement and their valuable comments. I am heartily thankful to Dr. James Reid for providing me with fungal cultures from his culture collections and also for his help, support, interest and valuable hints. I would like also to thank my advisor, my thesis supervision committee members as well as my external examiner Dr. William Hintz (University of Victoria) for their general and specific constructive comments that have greatly enhanced the value of this thesis. I would like to thank my former and present lab mates; Dr. Mahmood Iranpour, Dr. Jyothi Sethuraman, Dr. Sara-Taylor Mullineux, Shelly Rudski, Chen Shen, Iman Bilto and Michael Pogorzelec for supporting me in my research work, and I would like also to thank my awesome lab mate Tuhin Kumar Guha for helping in I-OmiI purification. I would like to express my deepest gratitude to all the faculty members and graduate students in the Department of Microbiology specially Dr. Linda Cameron, Dr. Chris Rathgeber, Aniel Moya-Torres and Munmun Nandi. Not to forget, great IV ________________________________________________________________________ appreciation goes to Sharon Berg, Madeleine Harris, Karen Hamill and Stephanie Moorhouse for continuous help and support during the project. I am indebted to many of my teachers and friends for shaping my personality and giving me the enthusiasm to complete this thesis and for everything, especially: Dr. Abdel-Wahed Moustafa, Dr. Akram Abu Seadah, Dr. Essam K. Ezz Eldin, Dr. Ahmed Abdel-Azeem, Dr. Moustafa Awad, Mr. Khaled Salah, Hossam Abdel Moniem, Belal Saleh, Tamer Saad, Ahmed Khalaf, Mohamed Fawzy, Alaaa Rashad, Mohamed Ali, Hatim Hemeda, Ezz-Eldin Mohamed and Mohamed Mostafa. Financial support of this project was provided by Discovery grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to Dr. G. Hausner and from the Egyptian Ministry of Higher Education and Scientific Research through the Bureau of the Egyptian cultural and Educational Affairs in Canada to M. Hafez. I also would like to acknowledge the financial support from the Faculty of Graduate Studies, the Faculty of Science and the Graduate Students’ Association at the University of Manitoba for supporting me financially to attend and present my thesis results in scientific conferences. Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the project and I would like to dedicate this work to my family, my parents, my siblings, my wife and my beloved daughters Nada and Mariam. V ________________________________________________________________________ TABLE OF CONTENTS Abstract …………………………………………………………………………… II Acknowledgements …………………………………………………………...….. V Table of contents …………………………………………………………………. VII List of tables ……………………………………………………………………… XV List of figures …………………………………………………………………….. XVI List of abbreviations ……………………………………………………………... XXII General introduction …………………………………………………………... 1 Chapter 1. Literature review ……………………………………………………. 5 1.1. Ophiostomatoid fungi ………………………………………………………… 5 1.2. Dutch elm disease and blue stain fungi ………………………………………. 6 1.3. Fungal mitochondrial genome ………………………………………………... 8 1.4. Mobile introns………………… ……………………………………………… 11 1.5. Group I introns ………………………………………………………………... 12 1.5.1. Distribution …………………………………………………………. 12 1.5.2. Structure …………………………………………………………….. 12 1.5.3. Splicing ……………………………………………………………... 16 1.5.4. Mobility …………………………………………………………….. 20 1.6. Group II introns ……………………………………………...……………….. 26 1.6.1. Distribution …………………………………………………………. 26 1.6.2. Structure …………………………………………………………….. 27 1.6.3. Splicing ……………………………………………………………... 32 1.6.4. Mobility …………………………………………………..………... 32 VI ________________________________________________________________________ 1.6.5. Evolution ……………………………………………………………. 35 1.7. Distribution of group I and II introns in rRNA genes ………………………… 39 1.8. Homing endonucleases ……………………………………………………….. 43 1.8.1. Nomenclature ……………………………………………………….. 44 1.8.2. Families of homing endonucleases …………………………………. 45 1.8.2.1. LHEases ……………..……………… …………………… 50 1.8.2.2. GIY-YIG HEases …………………………………………. 53 1.8.2.3. H-N-H HEases ……………...….…………………………. 54 1.8.2.4. His-Cys box HEases………………..……...……………… 55 1.9. Homing endonucleases and restriction endonucleases ……………………….. 55 1.10. Applications of homing endonucleases …………………………………… 57 1.10.1. Engineering homing endonucleases ……………………………….. 65 1.10.2. Future prospects …………………………………………………… 68 1.11. Research objectives ………………………………………………………….. 70 1.11.1. Screening for the presence of introns and IEPs in the rns gene of ophiostomatoid fungi ……………………………………………………… 70 1.11.2. Biochemical characterization of homing endonucleases encoded within group I and II introns ……………………………………………… 71 Chapter 2. General materials and methods …………………………………….. 72 2.1. Fungal strains and growth conditions ………………………………………… 72 2.2. DNA extraction ……………………………………………………………….. 72 2.3. PCR amplification …………………………………………………………….. 73 2.4. PCR products purification ………..…………………………………………... 80 2.5. Cloning of PCR products and purification of plasmid DNA ………………… 81 VII ________________________________________________________________________ 2.6. DNA sequencing ……………………………………………………………… 81 2.7. Sequence and phylogenetic analysis ………………………………………….. 82 2.8. Intron nomenclature and secondary structure modeling ……………………… 84 2.9. RNA extraction and Reverse Transcription-PCR (RT-PCR) ………………… 85 2.10. Ancestral state reconstruction ……………………………………………….. 85 2.11. Overexpression and purification of I-OmiII ………………………………… 86 2.11.1. Construction of the expression plasmid …………………………… 86 2.11.2. Overexpression of I-OmiII ………………………………………… 87 2.11.3. Purification of I-OmiII …………………………………………….. 92 2.12. In vitro endonuclease assay for I-OmiII ……………………………………. 93 2.13. Determination of the optimum temperature for I-OmiII ………………….. 94 2.14. I-OmiII cleavage site mapping ……………………………………………... 95 2.15. Overexpression and purification of I-OmiI ……………………………….. 96 Chapter 3. Characterization of the O.ul-mS952 intron: a potential molecular marker to distinguish between Ophiostoma ulmi and Ophiostoma