The ancient gene c12orf29 an exploration of its role in the chordate body plan

THE ANCIENT GENE C12ORF29: AN EXPLORATION OF ITS ROLE IN THE CHORDATE BODY PLAN Thor Einar Friis Bachelor of Science (Hons) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Health and Biomedical Innovation Faculty of Built Environment and Engineering Queensland University of Technology January 2013       Keywords   Complementary DNA library  Evolutionary biology  Developmental biology  Skeletal development  Cartilage  Phylogeny  Chordate body plan   The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan  i      ii    The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan    Abstract The sheep (Ovis aries) is commonly used as a large animal model in skeletal research Although the sheep genome has been sequenced there are still only a limited number of annotated mRNA sequences in public databases A complementary DNA (cDNA) library was constructed to provide a generic resource for further exploration of genes that are actively expressed in bone cells in sheep It was anticipated that the cDNA library would provide molecular tools for further research into the process of fracture repair and bone homeostasis, and add to the existing body of knowledge One of the hallmarks of cDNA libraries has been the identification of novel genes and in this library the full open reading frame of the gene C12orf29 was cloned and characterised This gene codes for a protein of unknown function with a molecular weight of 37 kDa A literature search showed that no previous studies had been conducted into the biological role of C12orf29, except for some bioinformatics studies that suggested a possible link with cancer Phylogenetic analyses revealed that C12orf29 had an ancient pedigree with a homologous gene found in some bacterial taxa This implied that the gene was present in the last common eukaryotic ancestor, thought to have existed more than billion years ago This notion was further supported by the fact that the gene is found in taxa belonging to the two major eukaryotic branches, bikonts and unikonts In the bikont supergroup a C12orf29-like gene was found in the single celled protist Naegleria gruberi, whereas in the unikont supergroup, encompassing the metazoa, the gene is universal to all chordate and, therefore, vertebrate species It appears to have been lost to the majority of cnidaria and protostomes taxa; however, C12orf29-like genes have been found in the cnidarian freshwater hydra and the protostome Pacific oyster The experimental data indicate that C12orf29 has a structural role in skeletal development and tissue homeostasis, whereas in silico analysis of the human C12orf29 promoter region suggests that its expression is potentially under the control of the NOTCH, WNT and TGF- developmental pathways, as well SOX9 and BAPX1; pathways that are all heavily involved in skeletogenesis Taken together, this investigation provides strong evidence that C12orf29 has a very important role in the chordate body plan, in early skeletal development, cartilage homeostasis, and also a possible link with spina bifida in humans The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan  iii    iv  The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan    Table of Contents  Keywords   i  Abstract   iii  Table of Contents   v  List of Figures  . viii  List of Tables   xii  List of Abbreviations   xiii  Statement of Original Authorship  . xxii  Acknowledgements   xxiii  CHAPTER 1:  INTRODUCTION   1  1.1  Background   1  1.2  Context  . 2  1.3  Purposes  . 3  1.4  Thesis Outline   4  CHAPTER 2:  LITERATURE REVIEW   7  2.1  A brief introduction to skeletal biology  . 7  2.2  Complementary DNA libraries and their utility in biological research  32  2.3  The sheep as an animal model   35  2.4  Summary and Implications   37  CHAPTER 3:  METHODS AND MATERIALS   41  3.1  Methodology and Research Design   41  3.1.1  Introduction   41  3.2  Construction of a cDNA library to identify genes expressed in cells derived from sheep bone 44  3.2.1  Cell culture   44  3.2.2  RNA extraction   45  3.2.3  cDNA library construction  . 47  3.2.4  PCR based methods for isolating full length cDNA clones   53  The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan  v    3.2.5  Library Screening: Method for amplification and isolation of cDNA from plasmid  libraries that require no hybridization (MACH)   53  3.2.6  Validation of cDNA library   57  3.3  C12orf29 – Characterization of a protein of unknown fuction   59  3.3.1  Subcloning C12orf29 cDNA into an epitope tagged expression vector   60  3.3.2  Validation of C12orf29 antibody specificity by western blot (WB) analysis   75  3.3.3  Immunofluorescent (IF) microscopy  . 83  3.3.4  Immunohistochemistry (IHC)   85  3.3.5  RT‐qPCR analysis of C12orf29 expression in sheep primary cells   88  3.3.6  WB analysis of C12orf29 protein expression in sheep primary cells   90  3.4  Bioinformatics analyses of C12orf29  . 91  3.4.1  Phylogenetic analyses   91  3.4.2  Molecular genetics analyses   94  CHAPTER 4:  RESULTS   99  4.1  Results – Introduction   99  4.2  cDNA library construction   100  4.2.1  Isolation of GAPDH cDNA clone using the MACH protocol   107  4.2.2  Validation of the cDNA library   111  4.3  C12orf29 – Experimental Results   117  4.3.1  Subcloning and epitope tagging the C12orf29 cDNA clone   117  4.3.2  Validating the specificity of the anti‐C12orf29 antibodies   119  4.3.3  Immunofluorescent microscopy   125  4.3.4  Immunohistochemistry   130  4.3.5  Real time quantitative PCR analysis   145  4.3.6  WB analysis of C12orf29 protein expression in sheep primary cells   147  4.4  C12orf29 – Bioinformatics analyses  . 149  4.4.1  Phylogenetic analyses   149  4.4.2  Molecular genetics analyses  . 156  CHAPTER 5:  ANALYSIS AND DISCUSSION  . 173  5.1  vi  Introduction   173  The Ancient Gene C12orf29:             An Exploration of its Role in the Chordate Body Plan    355  LOCUS DEFINITION JX534544 1862 bp mRNA linear MAM 31-OCT-2012 Ovis aries transforming growth factor-beta (TGF-beta 3) mRNA, partial cds ACCESSION JX534544 VERSION JX534544.1 GI:410066863 KEYWORDS SOURCE Ovis aries (sheep) ORGANISM Ovis aries Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Caprinae; Ovis REFERENCE (bases to 1862) AUTHORS Friis,T.E., Stephenson,S.A., Whitehead,J.P., Xiao,Y and Hutmacher,D.W TITLE PCR based method for isolating clones from a cDNA library in sheep JOURNAL Unpublished REFERENCE (bases to 1862) AUTHORS Friis,T.E., Stephenson,S.A., Whitehead,J.P., Xiao,Y and Hutmacher,D.W TITLE Direct Submission JOURNAL Submitted (25-AUG-2012) Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, Brisbane, QLD 4059, Australia FEATURES Location/Qualifiers source 1862 /organism="Ovis aries" /mol_type="mRNA" /db_xref="taxon:9940" gene Homo sapiens (Human) v2 GGRSRGGWARSVRGLSRAGDCFRASPRLDRAPLESTPSIRTACWAHHEALGLRAAENAVC VCDGGERGAFLQKGASDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKP APECWIPAKETEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHP DDSGLLEISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPSLK HNDLLSWFEDCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSRPVIINMNLNK CDSAFDIKCLFNHFLKIDNQKFVRLKDIIFDV >Pan troglodytes (Chimpanzee) MKRLGSVQRKIPCVFVTEVKEEPSSKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKPAPECWIP AKETEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPSLKHNDLVSW FEDCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSRPVIINMNLNKCDSAFDI KCLFNHFLKIDNQKFVRLKDIIFDV >Gorilla gorilla (Gorilla) MKRLGSVQRKIPCVFVTEVKEEPSSKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKPAPECWIP AKETEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPSLKHNDLVSW FEDCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSRPVIINMNLNKCDSAFDI KCLFNHFLKIDNQKFVRLKDIIFDV >Pongo abelii (Orangutan) MKRLGSVQRKMPCVFVTEVKEEPSSKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKENAKEFFWNVEEDFKPAPECWIP AKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELVGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPSLKHNDLLSW FEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSRPVIINMNLNKCDSAFDI KCLFNHFLKIDNQKFARLKDIIFDV >Nomascus leucogenys (Northern white-cheeked gibbon) MKRLGSVQRKMPCVFVTEVKEEPSSKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKLAPECWIP AKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPSLKHNDLLSW FEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSRPVIINMNLNKCDSAFDI KCLFNHFLKIDNQKFARLKDIIFDV 356  Appendices    357  >Macaca mulatta (Rhesus macaque) MKRLGSVQRKMPCVFVTEVKEEPSAKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKPAPECWIP AKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQVRNLPSLKHNDLLSW FEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKCDSAFDI RCLFNHFSKIDNQKFARLKDIIFDV >Macaca mulatta (Rhesus macaque) v2 GRSRGGWARSASGVDAAGDCCRASPQLDWALLESALSTRTACWAHHEALGLRAAENAVCV CDGGERGAFRQKGASDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKPA PECWIPAKEIEQINGNPVPDENGHIPDWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPD DSGLLEISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQVRNLPSLKH NDLLSWFEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKC DSAFDIRCLFNHFSKIDNQKFARLKDIIFDV > Macaca fascicularis (Crab-eating macaque) v2 GRSRGGWARSASGLDPAGDCCRASPQLDWALLESALSTRTACWAHHEALGLRAAENAVCV CDGGERGAFRQKGASDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKPA PECWIPAKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHAD DSGLLEITAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQVRNLPSLKH NDLLSWFEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKC DSAFDIRCLFNHFSKIDNQKFARLKDIIFDV >Callithrix jacchus (Common marmoset) MKRLGSVQRKMPCVFVTEVKQEPSAKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKGNPKEFFWNVEEDFKPAPECWIP AKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFGIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGNKKHPLHLLIPHGAFQIRNRPSLKHNHLLSW FEGCKEGKIEGIVWHCGDGCLIKVHRHHLGLCWPIPDTYMSSRPVIINMNLNKCHSAFDI KCLFNHFSKIDNQKFARLKDIIFDV >Papio anubis (Olive baboon) MKRLGSVQRKMPCVFVTEVKEEPSAKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKENPKEFFWNVEEDFKPAPECWIP AKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQVRNLPSLKHNDLLSW FEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKCDSTFDI RCLFNHFSKIDNQKFARLKDIIFDV >Saimiri boliviensis (Squirrel monkey) MKRLGSVQRKMPCVFVTEVKEEPSSKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQAEKRFKNFLHSKGNPKEFFWNVEEDFKPAPECWIP AKEIEQINGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGNKKHPLHLLIPHGAFQIRNLPSLKHNHLLSW FEGCKEGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSRPVIINMNLNKCDSAFDI KCLFNHFSKIDNQKFARLKDIIFEV >Otolemur garnettii (Small-eared Galago) MRRLGSVQRKVPCVFVTEVKEEPSAKREHQPFKVLATEAVSHKALDADIYSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQADKRFKNFLHSKDNSKEFFWNVEEDFKPAPECWIP AKEIEQLDGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLRHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQITNLPTLKHNDLLSW FEGCREGQIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSKPVIVNMNLNKYDYAFDN KCLFHHFLKLDNQKFSKLKDIIFDT Appendices  357    >Ovis aries (Sheep) MRRLGSVQRKMPCVFVTEVKEEPSTKREHQPFKVLATETVSHKALDADIYNAIPTEKVDG TCCYVTNYKGQPYLWARLDRKPNKLAEKRFKNFLHSKQNSKEFFWNVEEDFKPVPECWIP AKDIEQLNGNPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLRHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHSDLLSW FDGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKYEYAFDA KCLFNHFSKIDNQKFGRLKDIILNV >Bos taurus (Cow) MRRLGSVQRKMPCVFVTEVKEEPSTKREHQPFKVLATETLSHKALDADIHNAIPTEKVDG TCCYVTNYKGQPYLWARLDRKPNKLAEKRFKNFLHLKQNSKEFFWNVEEDFKPVPECWIP AKEIEQINGNPIPDENGHIPGWVPVEKNSKQHCWHSSVVDYESEIALVLRHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHSDLLSW FDGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKYEYAFDA KCLFNHFSKIDNQKFGRLKDIILTV >Sus scrofa (Pig) MRRLGSVQRKMPCVFVTEVKEEPSAKREHQPFKVLATETISHKALEADIYSAIPTEKVDG TCCYVTTYKGQPYLWARLDRKPNKLAEKRFKNFLHSKQNSKEFFWNVEEDFKPVPECWIP AKDTEQLNGSPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHSDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHNDLLSW FEGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYLNSKPVTINMNLNKYEYAFDA KCLFNYFSKIDNQKFSRLKDIVFSV >Tursiops truncatus (Bottlenose dolphin) MRRLGSVQRKMPCVFVTEVKEEPSTKREHQPFKVLATETISHKALDADIHSAIPTEKVDG TCCYVTTYKGQPYLWALLDRKHNKQAEKRFKIFLYLKENSKEFFWNVEEDFKPVPECWIP AKEIEQLNGNPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLRHHSDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHNDLLSW FEGCREGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNNYVYAFDA KCLFNHFSKIDNQKFGRLKDIILNV >Canis lupus familiaris (Dog) MRRLGSVQRKMPCVFVTEVKEEPSGKREQQPFKVLATETLSHKALDADIYSAIPTEKVDG TCCYITTYKGQPYLWARLDRKPNKLAEKRFKNFLHSKQNSKEFFWNVEEDFKPVPECWIP AKEIEQLNGNPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHNDLLSW FEGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYMNSKPVVINMNLNKYDHAFDT KCLFNLFSKIDNQKFGRLKDIILDV >Felis catus (Cat) MRRLGSVQRKMPCVFVTEVKEEPSTKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCYITNYKGQPYLWARLDRKPNKLAEKRFKNFLHSKQNSKEFFWNVEEDFKPVPECWIP AKEIEQLNGNPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEVALVLKHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHNDLLSW FEGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKYDHAFDT KSLFNLLSKIDNQKFGRLRDIILDV >Ailuropod melanoleuca (Panda) MRRLGSVQRKMPCVFVTEVKEEPSSKREHQPFKVLATETLSHQALDADIYSAIPTEKVDG TCCYITTYKGQPYLWARLDRKPNKLAEKRFKNFLHSKQNSKEFFWNVEEDFKPVPECWIP AKETEQLNGNPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEVALVLKHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHHDLLSW FEGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKYDHAFDT KCLFSRLSKIDNQKFGRLKDIILDV 358  Appendices    359  >Equus caballus (Horse) MKRLGSMQRKMPCLSVTEGKEEPSSKREQQPFKVLATETVNHKALDADIYSAIPTEKVDG TCCYITTYKGQPYLWARLDRKPNKLAEKRFKNFLHSKQNSKEFFWNVEEDFKPVPECWIP AKEIEQFNGNPMPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDPGLLE VSAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPSLKHNDLLSW FEGCREGKIEGIVWHCNDGCLIKVHRHHLGLCWPIPDTYMNSKPVIINMNLNKYDYAFDT KCLFNHFSKLDNQKFGRLKDIILDV >Desmodus rotundus (Vampire bat) MKRLGSVQRKIPCVFVTEVKEELSTKREHQPFKVLATETVSHKALDADIYSAIPTEKVDG TCCYVTTYKGQPYLWARLDRKPNKLAEKRFKNFRHSKQNSKEFVWNVEEDFKPVAECWVP AKEIEQLNGNPVPDENGHIPGWVPVEKSNKQHCWHSSVVDYELGTALVLKQHPDDPGLLE VSAVPLSGLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIHNLPTLKYKDLLSW FEGCREGKIEGVVWHCNDGCLIKVHRHHLGLCWPIPDTYLNSKPVIVNMNLNKHDYAFDT KCLFHHFLKIDNQKFSRLKDIILDV >Oryctolagus cuniculus (European rabbit) MRRLGSVQRKMPCVFVTEVKEEPSTKREHQPFKVLATETVSHKALDADIYSAIPTEKVDG TCCYVTTYKGQPYLWARLDRKPNKQADKRFKNFLHSKDNSKEFFWNIEEDFKPVPECWIP AKEIEQLNGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFEIALVLKHHPDDPGLLE ISAVPLADLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFQIRNLPTLKHNDLLSW FEGCREGKIEGIVWHCSDGCLIKVHRHHLGLCWPIPDPYMNSKPVVINMNLNKYDCAFDA KCLFNHFSKIDNQKFGRLKDIILDV >Loxodonta africana (African elephant) MRRLGSVQRKMPCVFVTEVKEEPSAKREHQPFKVLATETINHKALDADIYSAVPTEKVDG TCCYVTTYKGQPYLWARLDRKPNKQAEKRFKKFLYSKENSKEFFWNVEEDFKPVPECWIP AKEIEQLDGKPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFDIALVLKHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPLHLLIPHGAFHIRNLPTLKHNDLLSW FESCREGKIEGIVWHCNDGCLIKVHRHHLGLHWPIPDTFMNSKPVIINMDLNKYDYAFDS KCLFSYFSKIDNQKFSRLKDIMFDV >Rattus norvegicus (Rat) MNRLGSVQRKMPCVFVTEVKAEPSAKREHQPFKVLATETLSEKALDADVYNAVATEKVDG TCCYVTNYKGQPYLWARLDRKPNKQADKRFKKFLHSKENAKEFHWNTEEDFKPVPECWIP AKEIEKQNGKPVPDENGHIPGWVPVEKNSKQYCWHSSVVSYEFGIALVLRHHPDDPGVLE ISAVPLSELLEQTLELIGTNINGNPYGLGSKKYPLHFLTPHGAFQVRNLPTLKHNDLLSW FEGCREGQIEGIVWHCGDGCLIKVHRHHLGLCWPLPDTYMNSKPVIINMNLDKYDCAFDH QSLFNKFSKIDKQKFDRLKDITLDI >Mus musculus (Mouse) MKRLGSVQRKMPCVFVTEVKAEPSAKREHQPFKVLATETLSEKALDADVYNAVATEKVDG TCCYVTNYKGQPYLWARLDRKPNKQADKRFKKFLHSKESAKEFHWNTEEDFKPVPECWIP AKEIEKQNGKPVPDENGHIPGWVPVEKGSKQYCWHSSVVNYEFGIALVLRHHPDDPGVLE ISAVPLSELLEQTLELIGTSINGNPYGLGSKKSPLHFLTPHGAFQVRNLPTLKHNDLLSW FEDCREGQIEGIVWHCGDGCLIKVHRHHLGLCWPLPDTYMNSKPVIINMNLNLNNYDCAF DNQSLFNQFSKIDKQKFERLKDIILDV >Peromyscus maniculatus bairdii (Prairie deer mouse) MRRLGSVQRKMPCVFVTEVKAEPSAKREHQPFKVLATETLSEKALDADVYSAIATEKVDG TCCYVTNYKGQPYLWARLDRKPNKQADKRFKKFLHSKESSKEFLWNTEEDFKPVPECWIP AKEIEQQNGKPVPDENGHIPGWVPVEKNSKQYCWHSSVVNYESGIALVLRHDPDKPGLLE ISAVPLSELLEQTLELIGTNINGNPYGLGSKKYPLHFLIPHGAFQVRNVPALKHNDLLSW FEDCREGKVEGIVWHCGDGCLLKVHRHHLGLCWPLPDTYMNSKPVIINMNLNKYDCAF Appendices  359    >Cricetulus griseus (Chinese hamster) MSRLGSVQRKMPCVFVTEVKAEPSAKREHQPFKVLATETLSEKALDADVYNAIATEKVDG TCCYVTNYKGQPYLWARLDRKPNKQADKRFKKFLHSKDNSKAFLWNMEEDFKPVPECWIP AKEIEQQNGKPVPDENGHIPGWVPVEKNSKQYCWHSSVVNYEFGIALVLRHDPDEPGLLE ICAVPLSELLEQTLELIGTNINGNPYGLGSKKSPLHFLIPHGAFPVRNLPTLKHNDLLSW FEDCREGKIEGIVWHCGDGCLIKVHRHHLGLCWPLPDTYMNSKPVIVNMNLNKYDCAFDN ECLFNQFLKKDKQKFDRLKDIMLDV >Cavia porcellus (Domestic guinea pig) MRRLGSVQRKMPCVFVTEVKDEPSAKREHQPFTVLATETISHKALDADIHSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQADKRFKNFLRSQENSKEFLWNVEEDFKPVPECWIP AKEIEHLKGNPVPDENGHIPGWVPVEKSNKQYCWHSSVVNYEFEIALVLKHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGNKKHPIHLLIPHGAFQIRNLPKLKHNDLLSW FEGCREGKIEGIVWHCSDGCLIKVTTKVLVLSWHYYVPTAYNSPKNLSCIINMNLNKYGY AFDTKCLFNHFSKIDNQKFGRLKDIILDV >Nannospalax galili (Blind mole rat) CYVTTYKGQPYLWARLDRKPNKQADKRFKKFLHSKENSKEFLWNIEEDFKPIPECWIAAK EAEQHNGHPVPDENGHIPGWVPVERTSKQYCWHSSVVNYELGIALVLRHHPDDPGLLEIS SVPLSELLEQTLELIGTNINGNPYGLGSKKYPLHFLIPHGAFQVRNLPALKHNDLLSWFE GCREGKIEGIVWHCGDGCLIKVHRHHLGLCWPLPDTYMNSKPVIINMNLNKYDYTFDNKC LFNQFFKIDKQKFGRLKDIIFDV >Heterocephalus glaber (Naked mole rat) MRRLGSVQRKMPCVFVTEVKDEASAKREHQPFKVLATETISHKALDADIHSAIPTEKVDG TCCYVTTYKDQPYLWARLDRKPNKQADKRFKNFLCSKENSKEFLWNIEEDFKPVPECWIP AKEIKHLKGNPVPDENGHIPGWVPVEKSNKQYCWHSSVVNYEFEIALVLMHHPDDPGLLQ ISPVPLTDLLEQTLELIGTNINGNPYGLGNKKHPIHLLIPHGAFQIRNLPRLKHNDLLSW FEGCREGKIEGIVWHCSDGCLIKVHRHHLGLSWPIPDTYMNSKPVIINMNLNKYDYTFDT KCLFNYFSKIDNQKFGRLKDITLDV >Spermophilus lateralis (Ground squirrel) MRRLGSVQRKMPCVFVTEVKDEPSAKREHQPFKVLATETISHKALDADIYSAIPTEKVDG TCCFVTTYKDQPYLWARLDRKPNKKADKRFKNFLHSKENSKEFLWNVEEDFKPVPECWIP AKEIEQLNGNPVPDENGHIPGWVPVDKNNKQYCWHSSVVNYEFEIALVLKHHPDDPGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGNKKHPLHLLVPHGAFQISNLPTLKHSDLLSW >Monodelphis domestica (Brazilian opossum) MRRLGSVQRKVPCVFVTEVKDEPSVKREHQPFKVLATETINHKALDADIYNAIPTEKVDG TCCYVTTYKGQPYLWARLDRKPNKQAEKRFKRFLYSKENSKEFIWNVEEDFKSVPECWIP AKEIEQLNGNPVPDENGHIPGWVPVEKNNKQYCWHSSVVNYEFELALVLNHHADDSGLLE ISAVPLSDLLEQTLELIGTNINGNPYGLGSKKHPVHLLVPHGAFQIKNQPALKHHDLLSW FEGCREGRIEGIVWHCNDGCLIKVHRHHLGLRWPIPDTYMNSQPVIINMNRTRYDCAFDS KCLFNHFSKLDNQKFGRLKDIKLDV >Ornithorhynchus anatinus (Platypus) MNEGEFLVQLQEERVTRRQEPFKVLATETINHKALDADIYNAIPTEKVDGTCCYVTTYKG RPYLWARLDRKPNKQAEKRFKRFLYSKENTKEFVWNVEEDFKPVPERWIPAKEIEYLNGN PVPDENGHIPGWLPVEKNNKQYCWHSSVVNYEFETALVLNCHAGDPGLLEISAVPLSDLL EQTLELIGTNINANPYGLGSKKHPVHLLVPHGAFKIKSQPSLKHSDLLTWFESCREGKIE GIVWHCSDGCLIKVHRHHLGLRWPIPDTHMNSQPVVVNISSSKYGCDLDSKCLFSHFSKL DSQKFGSLRDIKLDG >Trichosurus vulpecular (Brushtail possum) GSRAHGLEAALEPSVKREHQPFKVLATETINYKALDADIYNAIPTEKVDGTCCYVTTYKG QPYLWARLDRKPNKQAEKRFKRFLYSKENSKEFIWNVEEDFKSVPESWIPAKDIEQLNGN 360  Appendices    361  PVPDENGHIPGWVPVEKNNKQYCWHSSVVSYEFELALVLNHHADDPGLLEISAVPLSDLL EQTLELIGTNINGNPYGLGSKKHPIHLLVPHGAFQIKNQPTLKHNDLLSWFXGCREEKLK A >Gallus gallus (Chicken) MSRRGAVQRKVPCLFVTEVKEEPSVKRERQPFKVLATETLSEKALEADIYNAVPTEKVDG TCCYVTTYKGQPYLWARLDRRPNKQAEKRFKRFLYSVEDCKEFIWNVEEDFKPVPDTWIP AKEIEFSNGNPLPDENGHMPGWVPVEKNSKQYCWHSSVVSYETEMALVLKHHADPGLLEI RPVSLSELLEQTLELIGTNINANPYGLGSKKQPIHLLVPHGAFEIKNPPTLKQSDIVSWF EGCSEGKVEGIVWHCRDGCLIKLHRHHLGLCWPLAETYLNSQPVVISFNRNDYDCDFGPK SLFHQFSKLDGQRFDRLKDIKFDD >Taeniopygia guttata (Zebra finch) MSRRGAVQRKVPCLFVTEVKDEPSAKRERQPFKVLATDTITGKALEADVHNAVPTEKVDG TCCYVTTYKGLPYLWARLDRKPTKQGEKRFKQFLYSLEDCKEFVWNVEEDFKPVPDTWIP AKDIEFSNGNPLPDENGHMPGWVPVEKNSKQYCWHSSVVNYEAGVALVLKHHADPGVLEI SPVPLSEILEQTLELIGTNINANPYGLGNKKHPVHLLVPHGAFEIKNPPALKQNDILSWF ETCMEGKVEGIVWHCADGCLIKIHRHHLGLPWPLAETYLNSQPVVISFNRTKCDYDFEPK SLFHHFSMLDGQRFDRLKDIKFDA >Meleagris gallopavo (Wild turkey) MPARRRSEGRRARSAAEVTANGFRLRPSPFKVLATETLNEKALEADIYNAIPTEKVDGTC CYITTYKGQPYLWARLDRRPNKQAEKRFKRFLYSVEDCKEFIWNVEEDFKPVPDTWIPAK EIEFSNGNPLPDENGHMPGWVPVEKNSKQYCWHTSVVNYEAEVALVLKHHADPGLLEIRP VSLSELLEQTLELIGTNINANPYGLGSKKQPIHLLVPHGAFEIKNPPTLKQSDILSWFEG CCEGKVEGIVWHCRDGCLIKLHRHHLGLCWPLAETYLNSQPVVISFNRNDYDCDFGPKSL FHQFSKLDGQRFDRLKDIKFDD >Lonchura striata (White-rumped munia) MSRRGAVQRKVPCLFVTEVKDEPSAKRERQPFKVLATDTITGKALEADVHNAVPTEKVDG TCCYVTTYKGLPYLWARLDRKPTKQGEKRFKQFLYSLEDCKEFVWNIEEDFKPVPDTWIP AKDIEFSNGNPLPDENGHMPGWVPVEKNSKQYCWHSSVVNYEAGVALVLKHHADPGLLEI SPVPLSEILEQTLELIGTNINANPYGLGNKKHPVHLLVPHGAFEIKNPPALKQNDILSWF ESCMEGKVEGIVWHCHDGCFNQAPSPSSWFTXGXLQKHT >Anolis carolinensis (Green anole lizard) MSLPLGSVQQKMPCLFVTEVREEPSAKREHQPFKVLATETINQKALDADIYNAVPTEKVD GTCCYITTYKGQPYLWARMDRKPNKQTEKRFKRFLYSADNSRGFTWNVEEDFRTVPESWI PAKEIERCNGKPLPDENGHIPGWVPVEKNSRQYCWHASVVDYEFELALILKHHTEEPGFL EICPVPLSTFSEHTLELIGTNINANPYGLGSKKHPVHLLVPHGIFQIKNAPALNHNDILT WLDGCKEGKIEGIVWHCADGNLIKLHRHHLGLSWPIADPHLISQPVVVNFSGIKYDYNFE PNTLFDYFSKLDGQRFNSLRNIASDL >Echis carinatus sochureki (Eastern saw-scaled viper) SAIPTEKVDGTCCYITTYKGHPYLWARLDRKPNKQAEKKFKQFMYSAEYSKGFTWNIEED FRSVPECWIPAKNIEYYNGKPFPDENGHIPGWVPVEKNSKLYCWHSSAVDYEYELALILK HHAEEPDLLEICPVPLIEIIEQTLELIGTNINANPYGLGNKKHPVHLLVLHGTFKIKNAP SINRNDILTWLDGCKEGKIEGIVWHCRGGNLIKLHRHHLGLSWPIADPSLTSKPVTVTFC RAKYEYNFEPKTLFHYFSNLDGHRFNSLRDIISDL >Ambystoma tigrinum (Tiger salamander) VFAQERESGQPYLWARLDRKPNKQAEKRFKKCVHSKQEKDFVWNVEDDFKPVPDNWIPAK EIHQCNGSPLPDEAGHIPGWVPVEKGNKQYCWHSSVINYEAGVALVLRPQAEDTDISEVS LVQLCDLLEQTLELIGTNINSNPYGLGSKK Appendices  361    >Rana catesbeiana (American bullfrog) GGLEDPESLEICLVHLTELLEQTLELVGTNINGNPYGIGNKKNPIHFLVPHGTFLIKNLP ALNHNSLISWFNHCQEGKVEGIVWHCKDGSLIKLHRHHLGLSWPLKDTYLNSKPVFIRMN LCKYEEASLCLAQFAKIDALRYDSLKDVLLD >Danio rerio (Zebra fish) MRRLGSVQQKIPCVFLTEVRDEPSRKRDCQQFQVVATENVSPAALASDVHCAAATEKVDG TCCYVTTFNGEPYLWARLDRKPTKQADKRFKKYQYSQKTCKGFVWNVNEDFREVPEFWMA AHRVQHENGHPVPDEHGHIPGWVPVDHTNKQYCWHSSVVNYGTGVALVLKTHGEDEGQLE IVSVPLADLMEQTLELIGTNVNGNPYGLGSKKHPVHVLVPHGVLRIRNPPAVEFQQICSW FQECQEGRVEGIVWHCDDGMLIKIHRHHLGLKWPVADTFLNTRPVVVHVDESDADPCASE KDLFKSFSSVNRQTFSSVRDIQFEP >Tetraodon nigroviridis (Puffer fish) MRRLGSVQQKMQCVFMIEVKEETSRKRDGQQYQVVATETVNPVALEANVDCAIATEKLDG TCCYVTTFEGRPHLWARLDRKPNKQAEKRFKKYQHSHRSCKGFTWNMEDDFKTVPEVWIP AHGVKQDNGHPLPDEHGHIPGWVPVEKNNKQYCWHSSVVDHNTGKALVLRPSADDADSLE IAAVPLGDLLEQTLELIGTNVNGNPYELGCKKQPVHCLVSHGSVGVRNPPPVDFQQLRLW FQDSPEGRVEGIVWHCIDGTLVKVHRHHLGLSWPDGHTRLGDRPLLVHVDTALDELSSSE DSFARFSKLNGHRFGRLRDVRFDSS >Ictalurus punctatus (Channel catfish) MRRLGSVQQKIPCVFLTEVKEEASRKRDGQNFQVVATDTVNPLAVESDIHRALATEKLDG TCCYVTLYRGEPYLWARLDRKPTKQANKKFKKYQYFQKTCIGFTWNVEEDFRTVPESWIP ARRVQYESGHPVPDVHGHIPGWVPVDSCNQQYCWHASVVDYDARAALVLRPNGEDEALLE IASVPLTELMEQALELIGTNVNGNPYGLGSKKNPLHVLVPHGILRIRNAPAVEYHQLRSW FQENDEGRVEGIVWHCNDGVLVKIHRHHLGLKWPDGDAFLNSRPVVVHVDQFPSDPDASA DSWKNLFAALAGLTGCRFSSIRDVQLEA >Esox lucius (Nothern pike) MRRLGSVQQKIPCVFLTEVKEEQSRKRESQQFQVVATENVNPIVLESNINSALATEKLDG TCCYVSMYKEQPYLWARLDRKPTKQARKRFKRHQCSYRSGKGFAWDVEEDFKTVPETWVP ALRVEHQNGQPVPDEHGHIPGFKVYLSNAPNNPAPSCTTKFL >Oncorhynchus mykiss (Rainbow trout) RKSAIMRRFGSVQQKISCVFLTEVKEEQSRKRECQQFQVVATENVNPIALESNIDCALAT EKLDGTCCYVSIYKGQSYLWARLDRKPTKQADKRFKKHQYSHKSYKGFTWNVKEDFKTVP ESWVPAHRVQHHNGQTCTR >Gadus morhua (Atlantic cod) RLSSVQQKVPCLFLTEVKQEQSRKRLGQPFQVIATEHLNPLALESNVESALATEKLDGTC CYVTYYEGQPYLWARLDRKPTKLADKSFKKYQHSHKSSKGFTWKVEEDFRTVPEAWIPAH GVQHHEGTPIPDDHGHIPGWVPVQTDNKQYCWHSSTVDYDAGVALVLRPGDQSPDLLELA LVPLVDLMEQTLELIGTNVNGNPYGMGAKKQPLHALVSHGSVGVGGPSPPPLDLQGLTSW FQESREGLEGIVWHCDDGTLVKVHRHHLGLGWPAGDCSFSSRPVEVRVDWTADHDYRLPT KDLFASLSRLDGQRFSRLQDV >Hippoglossus hippoglossus (Atlantic halibut) MGRLGSVQQKIPCVFVTEVKEEQSRKRESQQFQVVATEHMNPVALEADINCALATEKLDG TCCYVTLYKGQPYLWARLDRKPNKQAEKQFKKYQHSHRSSKGFTWNVEDDFKMVPETWIP AHRVKHHNGYPVPDDLGHIPGWVPVEKDNKQYCWHSSVLDYEVGVALVLRPKADDEDMLE IVAVPLADLMEQTLELIGTNVNGNPYGLGSKKQPVHFLVSHG >Perca flavescens (Yellow perch) MRRLGSVQQKIPCVFLTEVKEEQSRKRDCQQFQVVATENVNPVALEANIDCAFATEKLDG TCCYVTVYKGQPHLWARLDRKPNKQAEKRFKKYQHSHRSCKGFTWNVECDFKTVPEAWIP 362  Appendices    363  AHGVKHHSGRPVPDEHGHIPGWVPVEKDNKQYCWHSSVVDEGVGAVLVLRPSSDDEDLLE IAAVPLADLLEQTLELIGTNVNGNPYGLGNKKQPVHCLVSHGSVGIRNPPPVDFQQLCSW FQESPEGRVEGIVWHCNDGTLVKVHRH >Cynoglossus semilaevis (Tongue sole) MRRLGSVQHKVPCVFVTEVKEEQSRKRECQQFQVLASDRVNPVALEADVHNAVATEKLDG TCCYVTLYKGRPHLWARLDRKPNKQAEKRFKKYQHVHRSCKGFSWNLEEDFKSVPEQWIP AHRVKHDNGRPIPDQLGHIPGWVPVDKDNKQYCWHSSVVD >Dicentrarchus labrax (European seabass) MRRLGSVQQKIVCVFVTEVKEEQSRKRDCQVFQVAATENVNPIALEANVDCALATEKLDG TCCYVAVYKGQPYLWARLDRKPNKQAEKRFKKYQHSHRTCKGFTWNVEEDFKTVPETWIP AHRVKHHNGHPVPDEHGHIPGWVPVEKDNKQYCWHSSVVDYEVRATLVLRPSADNEDGLE IAAVPLADLLEQTLELIGTNVNGNPYGLGSKKQPVHCLVSHGSIQISNPPPVDFQRLCSW FQESPEGRVEGIV >Leucoraja erinacea (Little skate) MQRLGTVQQKIPCAFATELRDEPSTKRPGQSYRVLATGELSVKALAADIHRAIATEKVDG TCCYVAKYKDSPYLWARLDRKPTKQAEKRFRKFQSSKSGSEFTWNFGEDFRKVPEAWIPA HGVAQSEGCPAAR >Squalus acanthias (Spiny dogfish) MQRLGSVQQKIPCAFSTQVRDEPASKRANQSFRVLAIGELSPKALEADIHHAVATEKVDG TCCYVAMCKDLPYLWARLDRKLTKQAEKRFRKFQSSKTSTEFTWNLDDDFRTVADAWIPA HGVVQLKGRLQPDENGHIPGWVPVEKGSKPYCWHASTVNYETGSALVFEPCSEKIF >Oreochromis niloticus (Nile tilapia) MRRLGSVQQKIPCVFLTDVKEEQSRKRDCQQFQVVATENVNPVALEANVHSALATEKLDG TCCYVTVHKGQHYLWARLDRRPNKQTEKRFKKYQHSHKSCKGFTWNIEEDFKPVPETWIP AHGVKHHNGRPVPDEHGHIPGWVPVERDNKQYCWHSSVVDYNVGLALVLRPRRDNEDMLE ITSVPLAELQEQTLELIGTNVNGNPYGLGSKKQPVHCLVTHGSVPIRNPPPVDFQQLCSW FHENPDGRVEGIVWHCSDGTLIKVHRHHLGLKWPEVNTCLGNKPAAIRVDAYGSTDLFTS FVALNGHCFSRLQDIHFEL >Saccoglossus kowalevskii (Acorn worm) MPGSVQCKVPCVFKTRVIEEKSNKRQYQQYNVVASNELQDSAIEDSIEEATPTEKLDGTC VYVHEYKGRPWLWARLDRKPTKQADKHFKRYQAAKRAWMLNGMSGEEPMWNWNADKDFRT ARDSWIPAKGVPIINGTAQPDLYGHIPGWLPVESKDRQYCWHSSTFDVNYGIAVVLKSSN DGNQYSMEVTIVELSELLEQTLELIGTNINGNPYGLGCKRNPIHVLVPHGVIQFPSSIPI SYDELKLWFESSDEGKVEGIVWHCKNQQLYKLHRHHFGLPWPIEDVYFTSRPVAINMDLS RYECDFDSTSMFSVLSQFNGQRFEQLRNVPQDTQTGS >Saccoglossus kowalevskii (Acorn worm) v2 MFCDETRPSVLYGRQCSARKYTEDFKGLMGRSSPPVVFAVENPAANWDIALTMKWSVDSR ILYERYDTRMSFAEAAKKYDALVRPVGGNRVVWRIFNEKLIGKIEFVSHAHLISDSVKWV PVDPPPTLDPEYMSWEVGGGGVRTNHILDRMTIDNKFTIQVNRRLMRKFTSEYYDPSSDP YSVVALNDIRRSALDDNIEKAIPTEKFDGTCVYVHEYNGRPWLWARFERRPRTKCAAKKY DLKPVPDNWIPAKGVQIIDGKPQPGRFGHILGWVPVEKENRQYCWHSSTVDMARGIAMVL KPTDQGNQVTIVELSELLEQPLELIGTKINGNPYGLGCKTTPVHFLVPHAGNSSIVKRSF RLPLSYPLLKLWFEDFHEGRVEGIVWYCENQQLYKLNR >Branchiostoma floridae (Florida lancelet) MTDRVSVQEKIPCVFRTAVLQRQSSKRHDQSYHVGALNEVTTKALEANVRKAVASEKVDG TCVYIQEFRGKPWLWARHDVKPNKQAERRFKKFQSAHQAWEVGDKEGPEPTFTWNVGKDL KKPPEHWEPARGVRRIDGQLQPSSSGHVPGWVPVLKDSRSHCWHLASTDLDKGVALLLQR RKDDCNKTLEVLTVRLEELLGQTLELVGTNVNANPYGLGSKKRPVHLLVPHGALKFDTPP Appendices  363    PFDHEELADWFATPAGQVEGVVWYCDDGQLFKLHRHHLGLTWPVEEPSLTTLPVLITVDI GQYEWDFPESSQFYLFGRLKGQQFDSIRVVQWLDDN >Ciona intestinalis (Sea quirt) MAAHNKHCVQSKIDCLFETEVQQLPSNKRLGQNYRVVATKVVAQSAIESGIEHSVATEKL DGTCCYIDAYQGKLVLCARLDRKPNKSTEKKFKKFQNEKREWMINGGQGKEPKYEWKYPD DMKVVPPCWYPAHGVRREGGMVQPDGNGHVPGWVPISSDARQYCWHQNAIIDDRALVLLR NTDGLVVTTLPLSKLAGKTFELIGTNINGNPYGLGSKASPFHILVEHGSFQITNPPRLDY DVIKDWLSASGTIEGIVWHCDDGQMFKVHRHHLNLSWPIKKQEDCDVIMPLLCQIPVTID IEQSIVTKQTQISKLMKLNRKMFSSLVDILV >Ciona savignyi (Sea quirt) MQKSVTCSVQSKISCIFGTQVIEIPSKKRFGQNYKIVATKQISKQALASDVKFATVTEKI DGTCCCVKKYQGQICLWARFDRKPSKSAEKRFKKFQNQHREWINNGKQGEEPRFKWNYPD DMKVVPQNWVPADGVERTGVGLVPDANGHIPGWVPISSDPRQYCWHHCVVVAGDSILVLL RNNGGLKITTVHMSELEGKTLELIGTHVNGNPYGLGSKQSPFHILVEHGQILVNNPPVID YDDIKDWLNDKAPIEGIVWHCEDGNMFKIHRHHLGLEWPCDVTTPLLCQMPVTIEIEDSV MIQQSPLINKLFESETVFFKSLKDVLS >Hydra magnipapillata (Freshwater hydra) MYKNHVNGKIKCIFEIDIVQNGKDRQTVVAGEHLTESFSKCLKNHVLRPTIKLDGTSCYV AKFNGKSWLYARHDVKPNKMVSRKYKQYQKNYGLKKEGFVWNLETDYNFPKNWIAAHNNP FDGSMCSKPIPAENGHLVGWVPVDPSVRTHLWHLSAVDLIGGFFLCLNENAEDESLIAQI ALLDDYLGATFELIGTKVNGNPYDIGTSNFPVHFYVQHGELSLKNPPEISKKELSNWFVQ QKDGLVEGIVWHGPDGNMFKIHRHHLGLPWPVKNLPFSNRTVYIDCNGYACAENVLLNKI VIYSGKTFDKIKNLFNDEPYNFS >Naegleria gruberi (Amoeboflaggelate) MSQQATIDSSSSPANMYFPVQTKISCLFEIEFLEKEKKVLAVNRLYKKVVNLPTNSDDIR VNPTIKIDGTCCLVRQEKLFKRYDRKLNASGSKKNKQFQKEISQGTADTNILQFDISKDF KEGPKGWIPCDSIAHLFTEENNSNVVSSQHLVGWIPVDSNDAADKWHSSAIVSINNEPHA IMLIPYFDQNDEQAKFSVEFRKLSSLENQTLELIGSKINGNVYNFPANEKQHFLVPHGWA SYSWKASNAFLENVKELTVEKLKSWFEDESNIMSKSEGIVFHIMYKTEEGKDKEMLIKIH RHHLNLDWPLKGSAIPQFQYQLQWIEEVLRLKSENKL >Crassostrea gigas (Pacific oyster) MPLLTSRWKVAMFTIKSETFRVRFPTKWQANSIALKIMSYATLSNLCCLLVLVAILNIDR SEATLGGATCNIPTGSPGASTLCTSNKNGFCAVDTLDIDKKTVAGICICYPGFTGDKCQT ADPATAPGGTTGTTSSSKGTGNAVGALALGGLAALALSQLGGGGLGGGQFQGYKVVASDV LKNTALLSDVTRSIATEKLDGTCVFIAEFKGRPWLWARLDRKPNKAGDKRFKQYRSSLQK WEQSSQDLPKPSLEWDIEKDFKQVPEHWIPASDVPIVNGHPQPDQNGHTPGMLGGEGSMH SCKEKFEVEKSLIVLLM >Hahella chejuensis (-proteobacteria) MKKIKTLFVIDRHSHLATEQAVPESAWVLQGMGLATEKFDGSACLFRAGRLWKRYDRKLR KPFAAKLRAGRLATLEHAMFKSAPDGFTPCEAEPDAITGHWPGWLPVSKASPEDRWHCEA LEQAADTLIEGQTYELVGPKVQGNRYQLQRHALWAHGGKILTPPERLDFHSLRDWMAAQS MEGVVWHAPDGRMAKLRRKDFGFSW 364  Appendices    365  Appendix E – Statistical analysis  Wilcoxon signed rank test of RT‐qPCR results    580 MOB 580 MOB 580 MOB 580 MOB 580 MOB 580 MOB 580 MOB 580 MOB osteo cont osteo cont cont cont osteo osteo wk wk hr wk 72 hr hr 72 hr wk Sample value 95 102 103 110 111 127 226 250 Rank Control rank score 17 580 TOB 580 TOB 580 TOB 580 TOB 580 TOB 580 TOB 580 TOB 580 TOB osteo cont cont cont osteo cont osteo osteo hr wk 72 hr hr 72 hr wk wk wk Sample value 75 82 88 100 102 106 120 155 Rank Control rank score 559 MSC osteo 559 MSC cont 559 MSC cont 559 MSC osteo 559 MSC cont 559 MSC cont 559 MSC osteo 559 MSC osteo hr hr wk 72 hr 72 hr wk wk wk Rank 8 19 Osteo rank score 15 Sample value 104 119 139 143 161 171 207 329 Osteo rank score Control rank score 21 Osteo rank score 16 20 For n = 4, lower and upper tail at  = 0.025 is 10, 26 This supports the H1 hypothesis, that the gene expression of C12orf29 is greater in response to the osteogenic induction medium than control medium Appendices  365    Appendix F – Transcript from the Science  Show on ABC Radio National  Broadcast Saturday 02 June 2012  Robyn Williams: Our PhD this week is another example of chasing an idea that seems to  have no application yet. A gene that's been around forever, but what is it for? Here's Thor  Friis from the Queensland University of Technology.  Thor Friis: Thank you, Robyn, for having me on the program. In the first stage of my PhD I  constructed a cDNA library from cells derived from sheep bones. This is a bit like casting a  net over a population of genes that are active in a particular cell or tissue type and gives us  a snapshot of the molecular activities taking place. The library yielded a number of interest‐ ing genes. Today I'm going to talk about a particular gene that gives rise to a protein of un‐ known function with a molecular weight of 37 kDa. I shall refer to it here as PUF37.  As a molecular biologist, exploring the role of a gene of unknown function is the equivalent  of discovering a brand‐new species in the wild, which is very exciting indeed. Nature does‐ n't really give away her secrets, so how does one go about unravelling the role of a gene?  The first step I performed was searching through biological databases to identify those spe‐ cies that carried the gene and those species that do not, to see what this might tell us about  a possible biological role. This analysis revealed that my gene of interest was present in  every species that has a notochord at some stage during their development. These included  all the vertebrates, including humans, as well as three invertebrate species.  A notochord is a primitive backbone‐like structure which organises the dorsal body plan of  all species within the Chordate superphylum. The fact that all species with a notochord  carry the PUF37 gene suggests it had a specific role in the Chordate body plan which is pos‐ sibly, but not exclusively, linked to the notochord.  My gene of interest also turns out to have an ancient pedigree. I identified a PUF37 homo‐ logue in a single‐celled organism. This finding suggests that the gene has been around since  the emergence of the earliest metazoan organisms more than 1 billion years ago. But its  history goes back much further than that. It appears that the template for the metazoan  PUF37 can be found in certain bacterial species, which means it had its origin in the Pro‐ karyote kingdom 2.2 billion years ago. Such high degree of conservation suggests a very  important biological function.  The next step in the investigation was to determine the cells and tissues in which the gene  could be found. We found that it had a specific expression profile in the extracellular matrix  of joint cartilage. Cartilage is an avascular tissue, meaning it has no blood supply, so the  protein is therefore most likely secreted from cells embedded within the cartilage, or from  366  Appendices    367  cells within the underlying subchondral bone. Its presence within the extracellular matrix  suggests it has a structural role. PUF37 may therefore have an important role in cartilage as  well as in notochord.  Exploring the biological role of this gene will contribute to our understanding of evolution‐ ary biology, in particular the relationship between cartilage and the notochord. For exam‐ ple, is notochord a cartilaginous tissue, as some scientists would suggest? Or did cartilage in  fact appropriate certain molecules from the notochord, an issue which underpins questions  such as how vertebrates developed in the first place. I'm hoping to continue my post doc  exploring these questions.  Robyn Williams: Before you go Thor…  Thor Friis: Yes?  Robyn Williams: Your name is unusual for an Australian. Where does it come from?  Thor Friis: Well, my folks are Norwegians, so I'm first‐generation, but I grew up in Norway,  so hence the slight Norwegian accent.  Robyn Williams: Thor Friis at Queensland University of Technology. And who knows what  that gene may lead to.    Appendices  367    Appendix G – Conferences and publications  Conference Presentations Friis, T.E.; Xiao, Y.; Crawford, R.W.; Hutmacher, D.W. Constructing a cDNA library from Ovis  aries: capturing the osteogenic transcriptome. Poster presentation at IHBI Inspires Post‐ graduate Student Conference. 2009  Friis, Thor; Stephenson, Sally; Whitehead, Jon; Crawford, Ross; Xiao, Yin; Hutmacher, Diet‐ mar W. C12orf29: an ancient gene highly conserved across the chordate phylum plays a role  in mammalian bone biology. Oral presentation at IHBI Inspires Postgraduate Student Con‐ ference. 2011 (Runner up best oral presentation)  Publications Mao, X., H. Peng, J. Ling, T. Friis, A. K. Whittaker, R. Crawford and Y. Xiao. 2009. Enhanced  human bone marrow stromal cell affinity for modified poly(L‐lactide) surfaces by the  upregulation of adhesion molecular genes. Biomaterials 30 (36):6903‐11.  Chen, C. Y., T. He, X. L. Mao, T. E. Friis, R. H. Qin and Y. T. Jian. 2010. A novel xylene substi‐ tute for histotechnology and histochemistry. Biotechnic & histochemistry : official publica‐ tion of the Biological Stain Commission 85 (4):231‐40.  Fang, W., T. E. Friis, X. Long and Y. Xiao. 2010. Expression of chondromodulin‐1 in the tem‐ poromandibular joint condylar cartilage and disc. Journal of oral pathology & medicine : of‐ ficial publication of the International Association of Oral Pathologists and the American  Academy of Oral Pathology 39 (4):356‐60.  Chen, C., F. Ren, T. Lu, T. Friis, T. He, X. Zhang and Y. Jian. 2010. Involvement of salivary  glands in regulating the human nitrate and nitrite levels. Archives of oral biology 55 (9):613‐ 20.  Prasadam, I., T. Friis, W. Shi, S. van Gennip, R. Crawford and Y. Xiao. 2010. Osteoarthritic  cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signal‐ ling pathway involving ERK1/2. Bone 46 (1):226‐35.   Prasadam, I., S. van Gennip, T. Friis, W. Shi, R. Crawford and Y. Xiao. 2010. ERK‐1/2 and p38  in the regulation of hypertrophic changes of normal articular cartilage chondrocytes in‐ duced by osteoarthritic subchondral osteoblasts. Arthritis and rheumatism 62 (5):1349‐60.   Quent, V. M., D. Loessner, T. Friis, J. C. Reichert and D. W. Hutmacher. 2010. Discrepancies  between metabolic activity and DNA content as tool to assess cell proliferation in cancer re‐ search. J Cell Mol Med 14 (4):1003‐13.   Reichert, J. C., M. A. Woodruff, T. Friis, V. M. Quent, S. Gronthos, G. N. Duda, M. A. Schutz  and D. W. Hutmacher. 2010. Ovine bone‐ and marrow‐derived progenitor cells and their po‐ tential for scaffold‐based bone tissue engineering applications in vitro and in vivo. J Tissue  Eng Regen Med.   Wu, C., Y. Zhang, Y. Zhu, T. Friis and Y. Xiao. 2010. Structure‐property relationships of silk‐ modified mesoporous bioglass scaffolds. Biomaterials 31 (13):3429‐38.   10 Zhang, Y., C. Wu, T. Friis and Y. Xiao. 2010. The osteogenic properties of CaP/silk composite  scaffolds. Biomaterials 31 (10):2848‐56.   11 Vlacic‐Zischke, J., S. M. Hamlet, T. Friis, M. S. Tonetti and S. Ivanovski. 2011. The influence of  surface microroughness and hydrophilicity of titanium on the up‐regulation of  TGFbeta/BMP signalling in osteoblasts. Biomaterials 32 (3):665‐71.   368  Appendices    369  12 Wu, C., W. Fan, M. Gelinsky, Y. Xiao, J. Chang, T. Friis and G. Cuniberti. 2011. In situ prepara‐ tion and protein delivery of silicate‐alginate composite microspheres with core‐shell struc‐ ture. Journal of the Royal Society, Interface / the Royal Society 8 (65):1804‐14.   13 Wu, C., W. Fan, Y. Zhu, M. Gelinsky, J. Chang, G. Cuniberti, V. Albrecht, T. Friis and Y. Xiao.  2011. Multifunctional magnetic mesoporous bioactive glass scaffolds with a hierarchical  pore structure. Acta biomaterialia 7 (10):3563‐72.   Appendices  369  ...  Chordate body plan   The Ancient Gene C12orf29:              An Exploration of its Role in the Chordate Body Plan i      ii    The Ancient Gene C12orf29:              An Exploration of its Role in the Chordate Body Plan ... link with spina bifida in humans The Ancient Gene C12orf29:              An Exploration of its Role in the Chordate Body Plan iii    iv  The Ancient Gene C12orf29:              An Exploration of its Role in the Chordate Body Plan ... hedgehog The Ancient Gene C12orf29:              An Exploration of its Role in the Chordate Body Plan xix    SIBLING small integrin-binding ligand N-linked Glycoproteins SLIP Self-Ligation of Inverse