CHARACTERIZATION OF GROUP I INTRONS IN THE RIBOSOMAL RNA INTERNAL TRANSCRIBED SPACERS OF EIGHT ORDERS OF SHARKS Veena Patil 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 2011 Committee: Dr. Scott O. Rogers, Advisor Dr. Paul Morris Dr. Jeffrey Miner ii ABSTRACT Dr. Scott O. Rogers, Advisor Group I introns are self-splicing, ancient and abundant, being distributed sporadically among many phylogenetic lineages. This study is the first to report group I intron elements in the rDNA spacer regions (ITS1 and ITS2) of any organism and is the first report in any metazoan spacer region. Putative group I introns have been identified in ITS1 and ITS2 regions of all eight extant orders of sharks that are included in this study. The secondary structure prediction and analysis of these putative group I introns reveal that the introns have maintained all of the regions found in functional introns and thus are probably group I introns capable of splicing. Phylogenetic analysis indicates that these introns horizontally transferred prior to the evolution of all Orders of the Chondrichthyes, appearing first in ITS1 region. Since their first appearance, they have been evolving in parallel with shark species. iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my advisor Dr Scott O. Rogers for his guidance, inspiration, support and cooperation. He has been a wonderful person and a scientist to work with. He was always accessible and directed me to resources useful for my research. He is a very calm minded person who has always encouraged me to learn and progress in science. I am ever grateful to him for the invaluable knowledge I received from him. I am sincerely thankful to my committee members Dr. Paul Morris and Dr. Jeffrey Miner for their valuable feedback, guidance and support during my study. I profusely thank our collaborators Dr.Mahmood Shivji who sent us DNA samples of sharks for our analyses, and fellow collaborator Michael J.Stanhope. I am very thankful to our previous lab members Armeria Vicol and Nancy Walker who laid initial ground work for this project. I cannot forget the valuable help and cooperation of my lab members Caitlin Knowlton, Sammy Jumma, Farida Sidiq, Amal Abu Almakarem, and Katie Heilman. My special thanks to Yury Mikailovich Shtarkman for his consistent moral and technical support throughout my studies, and also for his timely help and suggestions. My sincere thanks to all my family members, my husband without whose consistent support and love it would not have been possible for me to come to BGSU and finish my studies. He has been very cooperative and helpful throughout my studies. I extend my deepest gratitude to my parents who gave me a very good education from my childhood that has enabled me to do this today. I am also thankful for my in-laws for their help and cooperation. iv I am thankful to Dr. Vipaporn Phuntumart and Dr. Kamau Mbuthia for all their help and cooperation during my study in BGSU. I am immensely thankful to Department of Biological Sciences, BGSU for giving me this opportunity and supporting me throughout my studies. I am extremely thankful to Dee Dee Wentland, Deb McLean, Louise Small, Chris Hess , and Linda Treeger for their help and cooperation during my study. I profusely thank my friends Chaitanya Kota, Vaishali, Uksha, Shivani, Rohit, Abilash, Prasad, Raja, and any other friends whose names I might have not recalled, for their help and cooperation during my study here in BG. Veena Patil v TABLE OF CONTENTS Page INTRODUCTION Sharks……………………………………………………………………………………….. 1 Ribosomal DNA (rDNA = rRNA gene locus)……………………………………………… 5 Phylogenetic studies………………………………………………………………………… 5 Introns and Evolution……………………………………………………………………….. 6 Group I introns……………………………………………………………………………… 7 MATERIALS AND METHODS DNA Extractions…………………………………………………………………………… 11 Amplification of ITS1 and ITS2 sequences from sharks…………………………………… 11 Constructing of plasmids for ITS1 and ITS2 sequencing and in vitro transcpition……….... 13 Blast search………………………………………………………………………………….. 14 Sequence alignment using Clustal W……………………………………………………….. 16 Phylogenetic Analysis and Secondary Structure Analysis………………………………….. 16 RESULTS Blast search results………………………………………………………………………….. 19 Phylogenetic analysis ………………………………………………………………………. 22 Secondary structure comparison……………………………………………………………. 23 DISCUSSION………………………………………………………………………………. 25 REFERENCES……………………………………………………………………………… 69 vi LIST OF FIGURES 1 Amplification of ITS1 and ITS2 sequences …………………………………………………. 35 2 Plasmid with PCR product insertion site for sequencing and transcription …………………. 36 3 Secondary structure the ITS2 intron from Lamna nasus …………………………………….. 37 4 Secondary structure the ITS2 intron from Hexanchus griseus … …………………………….. 39 5 Secondary structure the ITS2 intron from Heterodontus francisci …………………………... 41 6 Secondary structure the ITS2 intron from Squalus acanthias ……………………………….. 43 7 Secondary structure the ITS2 intron from Carcharhinus brevipinna ……………………….. 45 8 Secondary structure the ITS2 intron from Rhincodon typus …………………………………. 47 9 Secondary structure the ITS1 intron from Ginglymostoma cirratum ………………………… 49 10 Secondary structure the ITS1 intron from Sphyrna lewini …………………………………. 51 11 Secondary structure the ITS1 intron from Lamna nasus …………………………………… 53 12 Secondary structure the ITS1 intron from Isurus oxyrinchus ………………………………. 55 13 Secondary structure the ITS1 intron from Heterodontus fransici ………………………….. 57 14 Secondary structure the ITS1 intron from Hexanchus griseus ……………………………. 59 15 Unrooted most parsimonius tree of putative group I introns from both ITS1 and ITS2 regions………………………………………………………………………………………… 61 16 Rooted phylogram (most parsimonious) of putative introns from both ITS1 and ITS2 regions………………………………………………………………………………………… 63 17 Rooted phylogram (most parsimonious) of putative introns from both ITS1 and ITS2 regions………………………………………………………………………………………… 65 18 Most parsimonious tree of putative introns from both ITS1 and ITS2 regions showing representative structures for each clade………………………………………………………. 67 vii LIST OF TABLES: 1. List of all the taxa examined from the extant orders of sharks……………………......12 2. Primers used for the amplification of ITS1 and ITS2 from sharks………………….. 13 3. Primers used for sequencing ITS 1 and ITS2 regions of sharks……………………… 15 4. GenBank (NCBI) accession numbers for ITS1 and ITS2 intron sequences determined in this study……………………………………………………………………………….18 5. List of putative group I introns found in shark sequences previously deposited in GenBank (NCBI) from other studies………………………………………………….20 6. Mean and Standard deviation of length of P1 to P10 from the shark ITS 1 and ITS2 putative introns………………………………………………………………………..24 INTRODUCTION Sharks Sharks belong to the Class Chondrichthyes, Superorder Selachimorpha (Berman and Rotman, 1995), which have cartilaginous skeletons and highly streamlined bodies. They first appeared in the fossil record about 420 million years ago. There are about 440 known species of sharks to date. There are two subclasses, one of them being Elasmobranchi to which sharks belong along with skates and rays (the other Subclass is Holocephali or chimaeras. They are diverse in their physical appearance, distribution, diet, and habits. Their size range is wide from the smallest known species, lantern sharks ( Etmopterus pusillus ), having an average length of 17 cm, to the largest known species, whale sharks ( Rhinocodon typus ), which have an average length of 12 m. Sharks are found in all seas around the world. They are mostly restricted to marine habitats and are rarely found in fresh water, except for bull and liver sharks, which are found in both marine and fresh water environments (Allen 1999). Order Squatiniformes consists of one family and one genus with 16 species, including Squatina californica (Carrier et al 2004). They are mostly benthic, and are typically found in temperate coastal and tropical upper bathyal habitats (Compagno, 1984). Squatina californica (commonly known as Pacific angel shark, angel shark, California angel shark or Monkfish) is found in eastern Pacific Ocean from southeastern Alaska to the Gulf of California and Costa Rica to Southern Chile. Order Squaliformes consists of six families composed of small or medium sized sharks that are found in cold water (Compagno, 1999). The Genus Squalus first appeared in the Upper Cretaceous (Cappetta, 1987). Squalus acanthias (common names: spiny dogfish, piked dogfish, spotted spiny dogfish, and spur dogfish) is the most prominent member of the Family Squalidae . 1 It is used as a source of food for humans and pets, and for liver oil, fish meal, fertilizer, and leather products. Order Pristiophoriformes includes one family with 2 genera. The Genus Pristiophorus includes five species, of which two have been used for this study. The Japanese saw shark (Pristiophorus japonicus ) is found primarily in the northwest Pacific near Japan, Korea, and northern China. The shortnose saw shark ( Pristiophorus nudipinnis ) is found along the east coast of Australia. Pristiophorus fossils appear in the Upper Cretaceous and are very similar to the extant sharks (Cappetta, 1987). The Order Carcharhiniformes includes eight families (Compango 2001). The Family Carcharhinidae includes blue shark ( Prionace glauca ), bull shark ( Carcharhinus leucas ), black tip shark (C. limbatus ), spinner shark,