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Bursting behavior of transcription factors under
various stress conditions
Joshua Sanchez, Dr. Megan Mcclean*
Lewis-Sigler Institute for Integrative Genomics
Princeton University, New Jersey
Abstract
In the model organism Saccharomyces cerevisiae a transcription
factor called Crz1 exhibits a biological phenomenon known as bursting in
which Crz1 proteins localize in the nucleus before delocalizing to help up
regulate the production of mRNA for a period of time (Cai, et al 2008).
The mRNA produced codes for proteins important for the survival of the
cell. It is unknown if other transcription factors demonstrate a similar
behavior and which environmental stresses stimulate this response. A
library of GFP tagged transcription factors allows us to observe the
behavior of the proteins using an epifluorescence microscope. Over 200
transcription factors were monitored under various stress conditions to
observe which ones display this bursting behavior. We hope to understand
which transcription factors display bursting and under what
environmental stresses.
(Cai, L et al Frequency-modulated nuclear localization bursts coordinate gene regulation Nature 2008.)
Procedure
Science Alliance
Experiment
Calcium
GFP collection taken
from frozen stock using
sterile toothpicks and
placed into liquid
media.
Ca
In the seminal study by Cai, et al 2008 it was discovered that
Crz1 exhibits bursting in response to excess calcium. We use
calcium in this experiment as a control to verify Crz1's bursting
behavior as well as discover if other transcription factors might
respond to calcium.
Liquid culture
stamped onto
plate to grow
single colonies.
Academics
Community
Environment
Results
When CaCl2 was added we
found that only a few of the
transcription factors
responded by bursting. The
transcription factors that
responded to calcium are
Crz1 and Msn2.
When sorbital is added to the
solution it causes osmotic
shock to the yeast cells. The
transcription factors that were
found to exhibit bursting or
localize due to this stress are
Msn2 and Xbp1.
Sorbitol
Background
Transcription Factor (TF) : A protein that binds DNA at specific sites
where it can regulate transcription.
mRNA
Sample of colonies
taken from plate
and placed into
overnight culture.
Surviva
l
Protein
Polymerase
Ribosom
e
TF
Bursting - A biological phenomenon in which transcription factor proteins
localize in the nucleus for a period of time to regulate the production of
mRNA coding for survival proteins during times of stress.
Culture placed into
fresh media and
allowed to grow
overnight while
shaking in round
bottom 96 well plate.
100uL of cell culture
placed into glass
bottom plate and
placed onto stage of
automated
epifluorescence
microscope.
Green fluorescent protein (GFP): A protein composed of 238 amino acids
which shows bright green fluorescence when exposed to blue
light.. Derived from the jellyfish Aequorea victoria.
(Huh, W et al Global analysis of protein localization in budding yeast, Nature 425-669, 2003)
Starvation
C
SUGAR
Bursting - A biological phenomenon in which transcription factor proteins
centralize(concentrate) in the nucleus for a period of time to help up regulate the
production of mRNA coding for survival proteins during times of stress.
GFP tagged proteins- Almost every transcription factor that exists in the
yeast genome has been tagged with GFP and is widely available to study.
Adding sorbital to cells causes a hyperosmotic shock
and removes water from the cell. Transcription factors
may centralize in the nucleus as a reaction to this stress.
www.advancedaquarist.com/2005/12/aafeature2_album/RaskoffAequoria2_480.jpg
By adding all other components to our media except for the
primary energy source glucose, we hope to see if any
transcription factors respond by centralizing to help survive
the stress.
Cadmium
Stress is then placed
into each culture and
video is recorded
periodically for 4hrs.
Video is screened and
analyzed for bursting
and each sample is
recorded and notes
taken of interesting
observations.
By adding cadmium to
the cell solution at a high
concentration we found
that the transcription
factor Pdr1 reacted
strongly to this stress.
http://www.smsu.edu/CampusLife/StudentCenter/The%20Centers/candy.jpg
Conclusion
We found that the bursting phenomenon is a very rare event
and occurred in only 1 in 200 of the transcription factors we
screened in response to a single stress. There are many more
stress conditions that could be tested to find which
transcription factors respond in this fashion. In the future we
shall look more in depth at specific transcription factors and
also look at what causes this phenomenon to occur.
Acknowledgements
Cd
http://
By adding glucose free
media to the cell solution
we cause a drop in their
primary food source that
stresses the yeast. The
transcription factors that
appear to respond to this
stress are: Msn2
http://images-of-elements.com/cadmium2.jpg
By adding cadmium, which is a heavy metal, we hope to
stress the yeast and see which transcription factors react to a
cadmium saturated environment.
Principal Investigator: Ph.D. Megan Mcclean
Laboratory: Lewis-Sigler Institute for Integrative Genomics.
Princeton Molecular Biology SRI: Professor Alison Gammie, Director
Laura Gallagher-Katz, Associate Director
The summer program is supported by the: Howard Huges medical
institute, the department of Molecular Biology, and the Lewis-Sigler
Institute for Integrative Genomics.
Thanks also to: Dr. Adolfo Sanchez, Ricardo and Michelle Sanchez,
Eddie Cervantes, Prof. Rey Morales, Dr. Dale Clark, and Science
Alliance