Insights Into a Dinoflagellate Genome Through Expressed Sequence Tag Analysis

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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

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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

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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
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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)

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Subjects of the Research

• Study gene content
• Investigate dinoflagellate evolution
• Analyze DNA packaging

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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

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How Is EST Made?

• From mRNA by using special enzymes to convert it
  to cDNA (complementary DNA) - mRNA is very
  unstable outside a cell

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How Is EST Made?
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Application of ESTs

• Discovery of new genes
• Mapping of the genome
• Identification of coding regions

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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

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Alexandrium tamarense

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

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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 1311 hour lightdark cycle
• cDNA library obtained, 3 EST sequenced

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A. Tamarense Life Cycle
http//www.irishscientist.ie/2003/contents.asp?con
tentxml03p95.xmlcontentxslis03pages.xsl
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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)

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BLAST hits
Singletons
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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 dont have a
  typical polyA signal)
• GC content high codons 3rd position strongly
  biased towards 60.8 in the coding region, 57.6
  in UTRs
• Stop codon TGA favored over TAG TAA

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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)

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Codon Usage
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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)

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Summary Gene Content

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

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Dinoflagellate DNA

• Dont 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

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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

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

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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

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Results Histone HLPs

• HLP concentration low (proteinDNA ratio 110,
  eukaryotes 11) 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

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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

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Questions?