i EVOLUTIONARY INVESTIGATION OF GROUP I INTRONS IN NUCLEAR RIBOSOMAL INTERNAL TRANSCRIBED SPACERS IN NEOSELACHII Lizette Cooper A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of The requirements for the degree of MASTER OF SCIENCE December 2018 Committee: Scott Rogers, Advisor Paul Morris Vipaporn Phuntumart ii ©2018 Lizette Cooper All Rights Reserved iii ABSTRACT Scott Rogers, Advisor In an ongoing study of nuclear ribosomal DNA (rDNA) in fishes, unusually large (970 - 1418 bp) internal transcribed spacer (ITS1 and ITS2) regions were discovered in a wide diversity of members of the clade, Neoselachii (sharks, skates, and rays). This contrasts with the lengths for rDNA ITS regions in other eukaryotes, being larger by 30 to over 1000%. The additional segments of between 303 to 653 bp were due to insertions of single elements that have characteristics of group I introns, including conservation of structural and catalytic core regions. These spacer introns (spintrons) appear to be closest to the IC1 subgroup, although group I introns of any subtype have never been previously reported in the rRNA gene locus of any animal taxon. The aim for this study is to analyze the evolution of these spintrons. The current hypotheses are that these spintrons were inserted into an ancestor of Neoselachii and Batoidea, moved from one ITS region to the other, and then each evolved independently. iv I dedicate this thesis to all of my family and friends, especially, my parents, Jared Prange, Aaron Kuhman, my fraternity chapter, and my students, who have supported me through this arduous journey. v ACKNOWLEDGMENTS I acknowledge Mahmood Shivji for the acquisition of the shark muscle tissue utilized in this study and Scott Rogers who has provided the necessary DNA sequences. I also acknowledge all previous students who were working on this project before me, including Nancy Walker, Armeria Vicol, and Veena Prabhu. vi TABLE OF CONTENTS Page INTRODUCTION................................................................................................................. 1 Neoselachii and Batoidea .......................................................................................... 2 Identification of Introns ............................................................................................. 2 Group I Intron Structures.......................................................................................... 3 Hypothesis...................................................................................................... 3 Objectives....................................................................................................... 3 CHAPTER I. THE STUDY................................................................................................... 5 Materials and Methods............................................................................................... 5 DNA Extraction.............................................................................................. 5 PCR Amplification......................................................................................... 6 DNA Sequencing and Alignments................................................................. 6 Spintron Structure Analyses........................................................................... 7 Phylogenetic Analysis.................................................................................... 8 CHAPTER II. RESULTS...................................................................................................... 9 DNA Alignments ....................................................................................................... 9 Phylogenetic Analysis................................................................................................ 9 Spintron Structure Analyses....................................................................................... 10 CHAPTER III. DISCUSSION................................................................................................. 11 REFERENCES……………………………………………………………………………… 14 APPENDIX A. FIGURES ....... …………………………………………………………… 18 APPENDIX B. TABLE OF SPECIES AND ACCESSION NUMBERS FOR THE SPINTRONS STUDIED……………………………………………………… ......................................... 23 1 INTRODUCTION Group I introns interrupt expressed gene regions and are characterized by areas of conserved secondary structure and short sequences that are essential for splicing (Cech 1988, 1990; Cech and Bass 1986; Cech, Damberg, and Gutell 1994; Cech and Herschlag 1997; Jaeger, Michel, and Westof 1997; Lambowitz and Belfort 1993; Shinohara, LoBuglio, and Rogers 1993). While many group I introns are capable of self-splicing in vitro when provided with the proper pH and free guanosine, chaperone proteins in vivo accelerate the reaction significantly (Lambowitz and Belfort 1993). Group I introns are the most diverse class of introns. They have been divided into twelve subgroups (designated IA1, IA2, IA3, IB1, IB2, IB3, IB4, IC1, IC2, IC3, ID and IE) based on sequence and structural differences (Michel and Westof 1990). Despite the broad phylogenetic distribution of these elements in viruses and bacteria, as well as in the chloroplast, mitochondrial, and nuclear genomes of lower eukaryotes, group I introns have thus far been conspicuously absent from animal nuclear genomes (Cech 1988; Michel and Westof 1990; Gargas, DePriest, and Taylor 1995; Michel and Westof 1996; Beagley, Okada, and Wolstenholme 1996; Rogers et al. 1993). It has been reported that group I intron elements are present within the shark ITS1 and ITS2 regions of nuclear rDNA (ribosomal DNA). These group I introns are present in all seven extant Orders of Neoselachii (within Elasmobranchii) and Myliobatiformes of Batoidea (Douday et al. 2003). The presence of these group I introns are novel, because they interrupt non-genic spacer regions of the internal transcribed spacers (ITS), rather than expressed genic regions as in other organisms (Beagley, C.T. 1996). In light of this unusual location, they have been termed "spintrons" (for spacer introns (Scott Rogers)) to reflect a previously undocumented spacer insertion. This study serves to investigate the nature of these spintrons. 2 Neoselachii and Batoidea One of the aims of this study is to gain a better understanding of the evolutionary history of the spintrons present in the sharks of Neoselachii, but to do so, the evolutionary history of Neoselachii and Batoidea must first be described. The traditional evolutionary tree of sharks most often commences at the Chondrichthyes during the Cambrian period around 500 mya (million years ago) with the first major divergence of an ancestor of living sharks called Elasmobranchii emerging around 440 mya. The next event is the divergence of Neoselachii, comprised of seven extant Orders of sharks and one Order of skates and rays, which appeared around 270 mya (Long 1995, Capetta 1987, Martin 1995). However, a more recent phylogram study was done using mitochondrial DNA to explore the relatedness of Batoidea with the Orders of extant shark, and the data strongly supported shark monophyly and infers that Batoidea diverged earlier than Neoselachii (Douady, C. 2003). Another study observed the LSU and SSU rRNA genes from twenty-two elasmobranchs, two chimeras, and two bony fishes, which fortified the separation of Batoidea and Neoselachii (Winchell, C. 2004). The monophyly of sharks data shaped the hypotheses of this study. Identification of Introns It was previously observed that the ITS regions of sharks were quite large (Figure 1), and an investigation to explore these large ITS regions was conducted by using Mfold to analyze the secondary structures as described elsewhere (Shivji, Rogers, Stanhope 1996). Upon closer inspection, the large ITS regions was attributed to the presence of an intron. This intron’s structure was confirmed to be a group I intron by Nancy Walker and Scott Rogers in an unpublished study. 3 Group I Intron Structures Group I introns interrupt expressed gene regions and are characterized by areas of conserved secondary structure and short sequences that are essential for splicing (Cech 1988, 1990; Cech and Bass 1986; Cech, Damberg, and Gutell 1994; Cech and Herschlag 1997; Jaeger, Michel, and Westof 1997; Lambowitz and Belfort 1993; Shinohara, LoBuglio, and Rogers 1993). The secondary structure of a group I intron is folded by domains labeled P1-P10 as depicted in Figure 3. The P4-P5-P6 domain serves as a scaffold of group I introns to aid in lining up the exons that are included in P1 and P10 (Cech 1988). The P-9-P7-P3-P8 domain is the catalytic domain, and thus performs a vital function in intron splicing. P7 holds the free guanosine that initiates the first reaction of the two-reaction process of splicing (Cech 1988). To summarize, the discovery of these novel spintrons in the ITS regions of sharks encouraged a further investigation of spintron sequence morphology, structure analysis, and phylogenetic analyses. The following hypotheses were constructed to test. Hypothesis 1. The first appearance of these spintrons was seen in an ancestor to both Neoselachii and Batoidea. 2. The initial insertion of the spintron was into ITS1, and then a copy inserted into ITS2 in an early Elasmobranch lineage. 3. The spintrons in ITS1 and ITS2 evolved independently after the initial insertions. Objectives 1. Align the ITS regions of Neoselachii species from all seven extant Orders with Orders from Batoidea.