Chani & Malki present: Project adviser: Dr. Ron Wides The OdzFinder

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 Odz protein is expressed in neurons, developing brain and hindgut Odz protein is expressed in segmentation. Od O d z  Belongs to pair rule gene family  Plays a crucial role in the CNS during fetal development

The Odz Family Ten-m1 Ten-m2 Ten-m3 Ten-m4 Ten-a Ten-m Vertebrates Arthropods Odz gene orthologs have been found in 3 phylums: Nematodes

The Odz Protein  2731 Amino Acids III.hydrophobic sequences, probably transmembrane sequence EGF-like domainIntracellular kinase substrate domainODZ The only pair rule gene that encodes a protein!  Contains 3 domains: I. extracellular EGF-like repeats II. tyrosine kinase phosphorylation sites

EGF-like Repeats 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 EGF-like domain:  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

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 new EST libraries Data Bases Se/uences discovered in the lab EST Libraries Odz DataBase  Extracting DNA from various innocent creatures  BLASTing existing databases

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 We need a program to automatically extract Odz hits from NCBI Blast results!!! 2. Every day… New sequences are added to the existing databases New EST libraries are released

A perl program that will automatically extract Odz hits from NCBI Blast results. The OdzFinder

Blast Report Tax Report UpdateDatabase Combination Look up table Evalue>y? Score>x? Evalue>y? Odz EGF? Metazoan? Prokaryote? All EGF No EGF Mixed EGF no yes input S.O.F.T - screen Odz Flow Template

>gi| |gb|AC Apis mellifera BAC clone RP11-18D7, complete sequence Length = 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  The program extracts relevant information from each hit: input Blast Report  BLASTS are performed on the Odz orthologs  The results are sent to the OdzFinder program to be filtered.

>gi| |gb|AC Apis mellifera BAC clone RP11-18D7, complete sequence Length = 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 Taxonomy Report Eukaryota hits 41 orgs [root; cellular organisms]. Bilateria hits 33 orgs [Fungi/Metazoa group; Metazoa; Eumetazoa].. Coelomata hits 31 orgs... Deuterostomia hits 23 orgs.... Chordata hits 22 orgs..... Euteleostomi hits 21 orgs [Craniata; Vertebrata; Gnathostomata; Teleostomi] Tetrapoda hits 14 orgs [Sarcopterygii] Amniota hits 12 orgs Eutheria hits 10 orgs [Mammalia; Theria] Search for eukaryotic and metazoan results. Build prokaryotic database for possible future use. Evolutional distance becomes relevant when dealing with EGF-like repeats. 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. input Blast ReportTax Report ; rootroot; cellular organisms; Eukaryota; Fungi/Metazoa group; Metazoa; Eumetazoa; Bilateria; Coelomata; Protostomia; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Hymenoptera; Apocrita; Aculeata; Apoidea; Apidae; Apinae; Apini; Apiscellular organismsEukaryotaFungi/Metazoa groupMetazoa EumetazoaBilateriaCoelomataProtostomiaPanarthropodaArthropoda MandibulataPancrustaceaHexapodaInsectaDicondyliaPterygota NeopteraEndopterygotaHymenopteraApocritaAculeataApoideaApidae ApinaeApini Apis

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 : EGF? 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 Query Hit II. The hit is completely outside the query’s EGF-region Query Hit III. The hit is partially in the query’s EGF-region Query Hit

Get a better picture..

 score & e-value are examined  Set low threshholds to ensure that very small hits are not missed - some times they are translocations Position I : The hit is completely outside the query’s EGF-like region Evalue<y? Score>x? Odz yes No EGF

Position II : The hit is completely inside the query’s EGF-like region Look up table example: In order to prevent acceptance of non-odz hits with high scores due to their egf-region, a look up table was established evolutionally close query & subject high id % demanded evolutionally distant query & subject low id % demanded Odz ParalogOdz OrthologHitQuery 70%95%Homo SapiensMus Musculus 55%75%Drosophila Melanogaster Mus Musculus Look up table Score>x? Evalue>y? Odz yes ? All EGF

Position III : 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. AB Treat like II Is it more like A or like B? Treat like I Mixed EGF

DBI Update Database : Data flow through 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 DBD::MSQL MySQL RDBMS DBI Perl Script

speciesscoregi Xenopus Apis mellifera Gallus gallus Homo sapiens Rattus norvegicus Mus musculus Drosophila melanogaster Caenorhabditis elegans Gasterosteus aculeatus Results!

Special thanks to our project adviser Dr. Ron Wides For his guidance, patience & Krispy Kreme donuts