1 / 31

Insights Into a Dinoflagellate Genome Through Expressed Sequence Tag Analysis

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.

enye
Download Presentation

Insights Into a Dinoflagellate Genome Through Expressed Sequence Tag Analysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 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

  2. 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”

  3. 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

  4. 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)

  5. Subjects of the Research • Study gene content • Investigate dinoflagellate evolution • Analyze DNA packaging

  6. 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

  7. How Is EST Made? • From mRNA by using special enzymes to convert it to cDNA (complementary DNA) - mRNA is very unstable outside a cell

  8. How Is EST Made?

  9. Application of ESTs • Discovery of new genes • Mapping of the genome • Identification of coding regions

  10. 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

  11. Alexandrium tamarense • Toxic blooms • Shellfish poisoning • Peridinin-containing plastid • As a haploid (143 chromosomes)

  12. 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

  13. A. Tamarense Life Cycle http://www.irishscientist.ie/2003/contents.asp?contentxml=03p95.xml&contentxsl=is03pages.xsl

  14. 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)

  15. BLAST hits Singletons

  16. 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

  17. 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)

  18. Codon Usage

  19. 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)

  20. Summary: Gene Content • A. tamarense most closely related to Plasmodium falciparum (both members of alveolate linkage w/ dinoflagellates and apicomplexans)

  21. 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

  22. 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

  23. Alignment of A. tamarense H2A.X with Eukaryotic Homologs

  24. 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)

  25. Alignment of HLPs from Dinoflagellates (red) and Bacteria (blue) and HU Proteins from Bacteria (black) Proline • Bordetella petrussis (Bph2) – role in virulence gene expression & shares limited sequence w/ histone H1

  26. 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

  27. 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

  28. Questions?

More Related