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Transcript
Identifying the SMAD family of
transcription factors in the Cnidarian
model Aiptasia
By: Jennifer Osburn
Mentor: Dr. Virginia Weis
• 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
• 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
http://www.columbia.edu/itc/eeeb/baker/N0316/Lecture%202/
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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
http://www.uq.edu.au/news/images/media/DSCN0198-1.jpg
• Involved in immune system and regulatory functions (i.e. : induction of
tolerance)
• One of many cellular components homologous in higher organisms
• Homologs to TGF-β are present in the genome of Nematostella
vectensis, the only cnidarian genome sequenced to date
Type-I Receptor
TGF-b
ATP
Type-II Receptor
ATP
Bound SMAD protein
Serine/Threonine
Kinase receptor
ATP
Nuclear membrane
ATP
DNA
SMAD proteins bind to other SMADs
SMADs bind to DNA and
regulate gene expression
• 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
I predict that there will
be multiple SMAD
orthologs in Aiptasia
1. Identify and characterize SMAD orthologs from the
symbiotic anemone Aiptasia
2. Sequence the identified SMADs to enable further
research into the specific TGF-β proteins in the
anemone
3. Perform bioinformatics on the sequences in order to
build a phylogenetic tree of SMADs from Aiptasia and
other organisms
1. Design DNA primers using SMADs from Nematostella
2. PCR with these primers performed on cDNA from Aiptasia library
available in the lab in order to amplify SMADs
3. Run PCR product on a gel
4. Extract the desired bands from the gel and purify using a Qiagen kit
5. 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)
6. Sequence the resulting product at the CGRB
T7
Known Sequence
M13F
Protein Sequence
Plasmid
SP6
M13R
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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
Similar protein to SMADs
Found while looking at sequences
to create primers for SMADs
These bands are being cloned and
will be sent off for sequencing
Known Fragment
Successfully amplified the known
sequence and both forward and
reverse segments on a gel
Ladder
Facilitator protein used to bind SMADs
to DNA
Bands of Interest
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All segments of the sequence have been amplified but the primers have a
weak affinity for the DNA and seem to not work well
I originally used the primers
for M13 and later tried to
amplify using the T7 and SP6
regions
Currently, the known and
forward sequences are being
cloned and the reverse
sequence has been amplified
on a gel
Known Sequence
SP6
T7
M13R
M13F
Full Sequence
Plasmid
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
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
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
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