Transcript Insights Into a Dinoflagellate Genome

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
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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
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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?