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Insights Into a Dinoflagellate Genome Through Expressed Sequence Tag Analysis Jeremiah D. Hackett, Todd E. Scheetz, Hwan Su Yoon, Marcello B. Soares, Maria F. Bonaldo, Thomas L. Casavant, and Debashish Bhattacharya http://www.biomedcentral.com/1471-2164/6/80 Dinoflagellates • Marine producers & grazers of other bacterial & eukaryotic plankton • ~1/2 contain plastids, although many mixotrophic (food by photosynthesis & phagotrophy) • Many cause toxic “red tides” Red Tides • Result or more than 20 million cells/liter of seawater • Cause a variety of poisonings http://192.171.163.165/Edu_plankton_bio_indicators_of_change.htm Genetic Uniqueness of Dinoflagellates • Chromosomes are dense during the cell cycle except during DNA replication • Lack nucleosomes, DNA is associated w/ histone-like proteins (HLPs) • Crystal structure of DNA due its high concentration • Plastid genes located in minicircles w/ few genes per circle (most genes transferred to the nucleus) Subjects of the Research • Study gene content • Investigate dinoflagellate evolution • Analyze DNA packaging EST • Expressed Sequence Tags • A small piece of DNA sequence (200 – 500 nucleotides) • Used for sequencing of DNA that represent genes of interest • Can be generated from 5’ or 3’ end How Is EST Made? • From mRNA by using special enzymes to convert it to cDNA (complementary DNA) mRNA is very unstable outside a cell How Is EST Made? Application of ESTs • Discovery of new genes • Mapping of the genome • Identification of coding regions From cDNA to ESTs • Sequencing from 5’ or 3’ end • 5’ EST: sequencing the beginning portion of the cDNA, tends to be conserved across species (same gene family) • 3’ EST: sequencing the ending portion of cDNA, less crossed-species conservation Alexandrium tamarense • • • • Toxic blooms Shellfish poisoning Peridinin-containing plastid As a haploid (143 chromosomes) Library Construction • RNA extracted using Trizol (GibcoBRL) • RNA purified using Oligotex mRNA Midi Kit (Qiagen) • Culture strain produced by isolating a single cyst - a diploid in resting stage that produced haploid vegetative cells by meiosis • A single vegetative cell isolated • Grown at 20 oC on 13:11 hour light:dark cycle • cDNA library obtained, 3’ EST sequenced A. Tamarense Life Cycle http://www.irishscientist.ie/2003/contents.asp?contentxml=03p95.xml&contentxsl=is03pages.xsl Results: ESTs • • • • 6,723 unique ESTs (out of 11,171 3’ ESTs) Most clones were ~750 bp & singletons Largest cluster (46 ESTs) related to HLPs Second luciferin-binding protein (bioluminescence), photosynthetic proteins (Rubisco, light harvesting proteins) • One EST potentially coded for a protein w/ a plastid-targeting signal (candidate for dinoflagellate-specific protein) BLAST hits EST Processing • Each cluster was searched against SwissProt protein database using blastx • 515 hits had an e-value 10-20 but terminated within 10 aa of the SwissProt entry • 3’ UTRs had lengths btw 25-620 nt (avg ~155 nt) • 3’ UTRs lack a polyA signal (mechanism of polyadenylation happens different or don’t have a typical polyA signal) • G&C content high – codon’s 3rd position strongly biased towards: 60.8% in the coding region, 57.6% in UTRs • Stop codon TGA favored over TAG & TAA Summary: ESTs • Because only 20% of the significant hits to GenBank A. tamarense may be highly diverged and/or genes encode novel dinoflagellate-specific functions (or ESTs did not extend into the coding region of the transcript to be recognized) Codon Usage Results: Gene Content • BLAST showed that 609 out of 6,723 ESTs were comparable to P. falciparum (the most highly conserved proteins include many “housekeeping” proteins – a-tubulin or heat shock protein 70) • Evolutionary relationship but gene content substantially different (P. falciparum lost most genes related to plastid function or other metabolic genes) Summary: Gene Content • A. tamarense most closely related to Plasmodium falciparum (both members of alveolate linkage w/ dinoflagellates and apicomplexans) Dinoflagellate DNA • Don’t have nucleosomes but smooth chromosomal DNA strands, DNA is associated w/ HLPs • Chromosomes uniform in size, morphology, & remain condensed during the cell cycle & transcription from protruding loops Results: Histone & HLPs • Two rare ESTs out of 11,171 encode a partial histone H2A.X. One (169 aa) shares sequence identity to eukaryotic histone H2A.X (N-terminus longer than euk homologs but a-helices and histone fold conserved) • Comparison to Emilania huxlei – close relation to H2A (same monophyletic group) • Multiple origins of chromalveolates Alignment of A. tamarense H2A.X with Eukaryotic Homologs Results: Histone & HLPs • Alignment of the HLPs w/ other dinoflagellate HLPs & bacterial HU - moderate sequence similarity (bacterial HLPs have a longer N terminus, but secondary structure predictions are remarkably similar) (HU protein – histone-like DNA binding protein, necessary for protein-DNA assembly & DNA compaction) • Proline residue (*) not conserved thus unclear if able to interact w/ DNA as histones (bending DNA) Alignment of HLPs from Dinoflagellates (red) and Bacteria (blue) and HU Proteins from Bacteria (black) •Bordetella petrussis (Bph2) – role in virulence gene expression & shares limited sequence w/ histone H1 Results: Histone & HLPs • HLP concentration low (protein:DNA ratio = 1:10, eukaryotes 1:1) thus too low to function in DNA compaction - transcriptional regulators (role in repair of dsDNA that breaks non-homologous end-joining) • HLP gene maintained specifically for DNA repair & conserved for interaction with DNA as H2A • Similarities to HU proteins in structure due intracellular transfer from the mitochondrial or plastid endosymbionts Conclusion • The most extensive ESTs made & provided a useful glimpse into its nuclear genome • This data will be used for future research to understand the unique & complex cell biology & to understand toxin production • Future: - serial subtraction cDNA will be used to improve/maintain library - new cDNA libraries created under various growth conditions & life history stages to generate a more complex catalog Questions?