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Chani & Malki present: The OdzFinder Project adviser: Dr. Ron Wides WANTED Name: Odz a.k.a: Ten-m Family: pair-rule gene Length: 10,000 bp Getting to Know Odz … Discovered in D. Melanogaster in 1994 Belongs to pair rule gene family Plays a crucial role in the CNS during fetal development Odz protein is expressed in neurons, developing brain and hindgut Odz protein is expressed in segmentation. The Odz Family Odz gene orthologs have been found in 3 phylums: Vertebrates Ten-m1 Ten-m2 Ten-m3 Ten-m4 Ten-a Arthropods Nematodes Ten-m Ten-m The Odz Protein The only pair rule gene that encodes a protein! 2731 Amino Acids Contains 3 domains: I. extracellular EGF-like repeats II. tyrosine kinase phosphorylation sites III. hydrophobic sequences, probably transmembrane sequence EGF-like domain Intracellular kinase substrate domain ODZ EGF-like Repeats EGF-like domain: 30 - 40 amino acid residues Significant homology to epidermal growth factor (EGF) Has been found in single or multiple copies in a number of other proteins Generally found in the extracellular domain of membrane proteins or secreted proteins Involved in receptor-ligand interactions Includes 6 conserved cysteine residues involved in disulfide bonds x(4)-C-x(0,48)-C-x(3,12)-C-x(1,70)-C-x(1,6)-C-x(2)-G-a-x(0,21)-G-x(2)-C-x The lab’s goals: Genomics: To find a broad family of Odz gene Phylogenetic trees to discover segmentation mechanism Massive alignment to find conserved regions Biological in-vivo experiments to change regions Proteomics: The protein’s role How the protein functions The protein’s interactions with other proteins ( i.e : notch) Finding Odz Genes BLASTing existing databases Data Bases BLASTing new EST libraries Extracting DNA from various innocent creatures Odz DataBase EST Libraries Se/uences discovered in the lab Odz Database The collected data was organized by Michal Markovitz in a relational database. The database consists of 10 different tables. For example: 2 problems remained: 1. Blast results include many non Odz hits: • prokaryotic hits • non-metazoan hits • EGF region hits • Low similarity 2. Every day… • New sequences are added to the existing databases • New EST libraries are released 80 70 60 50 40 30 20 low score prokaryotic non-metazoan Odz Egf-like 10 0 We need a program to automatically extract Odz hits from NCBI Blast results!!! The OdzFinder A perl program that will automatically extract Odz hits from NCBI Blast results. S.O.F.T - screen Odz Flow Template Blast Report input Tax Report Prokaryote? no Metazoan? yes No EGF EGF? All EGF Mixed EGF Score>x? yes Score>x? Combination Evalue>y? yes Evalue>y? yes yes Odz UpdateDatabase Look up table Blast Report input BLASTS are performed on the Odz orthologs The results are sent to the OdzFinder program to be filtered. The program extracts relevant information from each hit: >gi|163076235|gb|AC765764.7 Apis mellifera BAC clone RP11-18D7 , complete sequence Length = 184032 Score = 153 bits (328), Expect = 3e-36 Identities = 59/59 (100%), Positives = 59/59 (100%) Frame = +3 / +3 Query: 3 IQHKTFKFHGNYIKQRFHPRIYK*RYKYQRFHPRIYK*NLNLYRVCCSHIILECLQTAH 179 IQHKTFKFHGNYIKQRFHPRIYK*RYKYQRFHPRIYK*NLNLYRVCCSHIILECLQTAH Subjct: 3 IQHKTFKFHGNYIKQRFHPRIYK*RYKYQRFHPRIYK*NLNLYRVCCSHIILECLQTAH 179 Blast Report input Tax Report Search for eukaryotic and metazoan results. Build prokaryotic database for possible future use. Evolutional distance becomes relevant when dealing with EGF-like repeats. >gi|163076235|gb|AC765764.7 Apis mellifera BAC clone RP11-18D7 , complete sequence Taxonomy Report Eukaryota .................................. 2502 hits 41 orgs [root; cellular organisms] Length = 184032 . Bilateria ................................ 2421 hits 33 orgs [Fungi/Metazoa group; Metazoa; Eumetazoa] root; cellular organisms; Eukaryota; Fungi/Metazoa group; Metazoa; = 153 bits (328), Expect = 3e-36 Identities = 59/59 (100%), Positives = 59/59 (100%) .Score . Coelomata .............................. 2396 hits 31 orgs Eumetazoa; Bilateria; Coelomata; Protostomia; Panarthropoda; Arthropoda; . . . Deuterostomia ........................ 2322 hits 23 orgs Frame = +3 / +3 . . . . Chordata ........................... 2296 hits 22 orgs Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Query: 3 IQHKTFKFHGNYIKQRFHPRIYK*RYKYQRFHPRIYK*NLNLYRVCCSHIILECLQTAH . . . . . Euteleostomi ..................... 2236 hits 21 orgs [Craniata; Vertebrata; Gnathostomata;179 Teleostomi] Neoptera; Endopterygota; Hymenoptera; Apocrita; Aculeata; Apoidea; Apidae; . . . . . . Tetrapoda ...................... 2022 hits 14 orgs [Sarcopterygii] IQHKTFKFHGNYIKQRFHPRIYK*RYKYQRFHPRIYK*NLNLYRVCCSHIILECLQTAH . . . . . . . Amniota ...................... 1908 hits 12 orgs Apinae; Apini;................... 1634 hits 10 orgs [Mammalia; Theria] IQHKTFKFHGNYIKQRFHPRIYK*RYKYQRFHPRIYK*NLNLYRVCCSHIILECLQTAH 179 .Subjct: . . . . . .3. Eutheria Apis The program will receive the BLAST hit’s Taxonomy Report and manipulate it into a manageable hash table. A default Taxonomy Report will be available when BLASTing against ESTs. EGF? Tenascin-m (odz) includes 8 EGF-like repeats The conserved EGF region gave problematic results. Many hits appear only due to their similarity to the EGF region. Query : Subject : High score!!! There are three possible positions regarding the hit’s relation to the query’s EGF-like region - I. The hit is completely inside the query’s EGF-region 525 804 2750 Query Hit II. The hit is completely outside the query’s EGF-region Query 525 804 Hit III. The hit is partially in the query’s EGF-region Query Hit 525 804 Get a better picture .. Position I : No EGF The hit is completely outside the query’s EGF-like region score & e-value are examined Set low threshholds to ensure that very small hits are not missed - some times they are translocations Score>x? yes Evalue<y? yes Odz Position II : All EGF The hit is completely inside the query’s EGF-like region In order to prevent acceptance of non-odz hits with high scores due to their egf-region , a look up table was established Score>x? yes evolutionally close query & subject evolutionally distant query & subject high id % demanded low id % demanded Evalue>y? yes Look up table ? Look up table example: Odz Query Hit Odz Ortholog Odz Paralog Mus Musculus Homo Sapiens 95% 70% Mus Musculus Drosophila Melanogaster 75% 55% Position III : Mixed EGF The hit is partially inside the query’s EGF-like region 2 Possibilities: A. False call ! An EGF hit with insignificant similarity outside of EGF-domains. B. The Real Thing ! EGF with adjacent regions of significant similarity. Is it more like A or like B? A Treat like II B Treat like I Update Database DBI A database interface module for Perl Enables Perl applications to access multiple database types Provides a consistent database interface independent of the actual database being used :Data flow through DBI Perl Script DBI DBD::MSQL MySQL RDBMS Results! gi score species 49256537 140 Xenopus 48096180 637 Apis mellifera 45382362 619 Gallus gallus 42658224 125 Homo sapiens 34932761 384 Rattus norvegicus 38087011 463 Mus musculus 45446084 419 Drosophila melanogaster 32565715 1604 Caenorhabditis elegans 41469033 760 Gasterosteus aculeatus Odz Odz not Metazoa Metazoa not EGF EGF Prokaryotic Prokaryotic Special thanks to our project adviser Dr. Ron Wides For his guidance, patience & Krispy Kreme donuts