Identifying the SMAD family of transcription factors in the Cnidarian model Aiptasia

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Identifying the SMAD family of transcription
factors in the Cnidarian model Aiptasia
By Jennifer Osburn Mentor Dr. Virginia Weis
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Coral Reefs

• Very important economically
• Tourism
• Food
• Play a large role in the carbon cycle
• Provide habitat for a large
• diversity of organisms
• Represent a huge source of bioactive molecules
  with applications in research or medicine

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Endosymbiosis

• Coral polyps house unicellular symbiotic algae
  in their endodermis within vacuoles
• The polyps provide nutrients and a safe living
  space for the algae and the algae provide carbon
  products to their host
• There are complex mechanisms in which the coral
  and symbiont interact with each other
• Cell signaling allows each partner to
  communicate
• and provide feedback to other cells

Cnidarian Host Cell
Nucleus
Algal Symbiont
Cell-Cell Communication
Nitrogen, CO2
Carbon Products
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re202/
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Bleaching

• Bleaching is a process in which the coral
  expels its symbiotic algae
• Due to a breakdown of tolerance of the symbiont
• Occurs when the organism is stressed due to its
  environment
• Most of the time this results in a decrease in
  coral health and often death
• Without symbiosis, calcareous skeleton cannot
  be formed

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The TGF-ß Pathway

• Involved in immune system and regulatory
  functions (i.e. induction of tolerance)
• One of many cellular components homologous in
  higher organisms
• Homologs to TGF-ß are present in the genome of
  Nematostella vectensis, the only cnidarian genome
  sequenced to date

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SMADs

• They serve as the structures that translocate
  between the cytoplasm and nucleus
• Purpose is to convert a signal into gene
  expression and regulation
• Three types of SMADs include receptor-regulated,
  inhibitory, and common-partner SMADs
• Multiple orthologs, homologous proteins between
  organisms, have been found
• 7 SMADs have been identified in the
  well-studied Nematostella anemone

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Prediction
I predict that there will be multiple SMAD
orthologs in Aiptasia
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What I planned

• Identify and characterize SMAD orthologs from the
  symbiotic anemone Aiptasia
• Sequence the identified SMADs to enable further
  research into the specific TGF-ß proteins in the
  anemone
• Perform bioinformatics on the sequences in order
  to build a phylogenetic tree of SMADs from
  Aiptasia and other organisms

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The method used

• Design DNA primers using SMADs from Nematostella
• PCR with these primers performed on cDNA from
  Aiptasia library available in the lab in order to
  amplify SMADs
• Run PCR product on a gel
• Extract the desired bands from the gel and purify
  using a Qiagen kit
• Clone purified products into the pGEM-T easy
  vector system
• (Ligation of the DNA into a plasmid and
  transformation for bacteria to take up the
  plasmid)
• Sequence the resulting product at the CGRB

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The cDNA library
SP6
Known Sequence
T7
M13R
M13F
Protein Sequence
Plasmid
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SMAD1-5

• Successfully cloned despite numerous
  complications with gel and buffers
• Two colonies were grown overnight in SOC medium
• These colonies were sent to CGRB for sequencing
• Sequences have been analyzed and BLASTed
  against other sequences
• BLASTing is a comparison of numerous sequences
  in a database

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SARA Protein
Similar protein to SMADs
Found while looking at sequences to create
primers for SMADs
Bands of Interest
Facilitator protein used to bind SMADs to DNA
Ladder
Known Fragment
Successfully amplified the known sequence and
both forward and reverse segments on a gel

-
These bands are being cloned and will be sent off
for sequencing
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SMAD2
All segments of the sequence have been amplified
but the primers have a weak affinity for the DNA
and seem to not work well
Known Sequence
I originally used the primers for M13 and later
tried to amplify using the T7 and SP6 regions
SP6
T7
M13R
M13F
Full Sequence
Currently, the known and forward sequences are
being cloned and the reverse sequence has been
amplified on a gel
Plasmid
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SMAD 6-7
While designing primers for SMADs 6 and 7, it
became apparent that they both have similar
sequences of similar lengths It is impossible to
distinguish them from each other on a gel or
during the cloning process I used the same
primers for both SMADs and once sequenced, may be
able to tell them apart SMAD 6-7 has been
amplified, purified, and is currently in the
process of being cloned for sequencing
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Complications
SARA and SMADs 2, 6, and 7 have all been cloned
previously but there have been problems with
bacterial colony growth The result is that the
sequencing has failed and the inserted fragment
into the bacteria is not the DNA I am searching
for The suspected problem is that the LB media
used for the bacteria was improperly made
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Conclusions
I have learned the process that I am following
for the summer as shown to me by Dr. Olivier
Detournay. I will continue my work with the
SMADs during the academic year and hope to have
complete sequences for all of the SMADs and SARA
found Other primers are being designed for use
in finding SMADs 3 and 4
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Acknowledgements

• Thank you to
• The Howard Hughes Medical Institute for
  allowing me this opportunity to take part in this
  summer research program
• Dr. Kevin Ahern
• The Weis Lab
• Dr. Virginia Weis
• Dr. Olivier Detournay
• Christy Schnitzler
• Wendy Phillips
• Emilie Dicks