GCAT-SEEKquence
The Genome Consortium for Active Teaching
NextGen Sequencing Group
NextGen Sequencing Request Form
Complete fields below, save file with your last name at the beginning of
the filename (e.g. newman-GCAT-SEEK Sequence request form.pdf) and
email to Vincent Buonaccorsi <BUONACCORSI@juniata.edu>
A. Contact Information
1. Name:
Irene M. Evans
2. Department: School of Life Sciences
3. Institution: Rochester Institute of Technology
4. Phone Number: (585) 385-3893
5. Email Address: IMESBI@RIT.EDU
B. Project Information
1. Title: What effect does a prion protein have on a yeast cell grown under stressful conditions?
2. Category:Transcriptomics
3. Total amount of sequence requested: Sequence enough to determine the transcriptional profile of
two yeast strains [PSI+] and [psi-].
4. Preferred technology: To be determined.
5. Do you have funds for a partial run next Spring? No
6.
Describe the background, hypotheses and specific aims (500 words max)
Background: Approximately a dozen yeast prion proteins have been described, but there is little information on
what role they play in a yeast cell. Our laboratory is studying the [PSI+] prion protein. The Sup35 gene product
can exist in the prion state [PSI+] or non-prion “normal” state [psi-]. The Sup35 gene product codes for a
translational release factor (eRF3) and in its prion state, eRF3 is inactive and allows read-through of stop codons.
Thus the translational profile may be altered and may alter the transcriptional profile. What role, if any, this
misreading of a mRNA containing a stop codon has on a cell is largely unknown.
We are asking whether the presence of a prion protein in the prion-positive [PSI+] yeast strain changes the
transcriptional profile of the cell. Our initial studies growing the [PSI+] and [psi-] yeast strains in rich YPD media
did not detect large (2x) variations in mRNAs. More recent studies show that two-fold transcriptional changes
are present when the yeast are grown in stressful media due to a poor nitrogen source. These initial studies
were done using spotted microarrays provided by GCAT.
We propose to continue these studies, but to switch to direct sequencing of the mRNAs using Next Generation
sequencing. Irene Evans is a member of GCAT-SEEK and plans to learn the NEXT-Gen Sequencing technology
and then proceed to use this technology to carry out the proposed project.
Hypotheses: Our hypothesis is that prion proteins allow a cell to survive in stressful conditions. We propose
that a cell changes its transcriptional profile differently if that cell contains a prion protein and is therefore
[PSI+].
Specific Aims:
1. To study the transcriptional profile of [PSI+] and [psi-] yeast cells growing in stressful media in order to
elucidate transcriptional differences between the strains.
2. To compare the transcriptional profile of a Sup35 mutant with the transcriptional profile of [PSI+] and [psi] strains to see if some of the transcriptional variations found are due to decreased Sup35 activity in
[PSI+].
C. Describe the methods [sample prep, calculation of amount of sequence required, analysis plan]
Total RNA will be isolated from the yeast strains using the Ambion Ribo-PURE Yeast kit. The purity of the
RNA will be checked by running the sample out on a denaturing agarose gel and looking at the tightness of
the rRNA bands. Protocols for mRNA and data analysis will follow best practices of the network.
D. Describe the role and number of undergraduates involved in the project, and how they would benefit.
I have 11 undergraduates in my Research group who are currently doing and learning microarray
technologies. These students are very excited about learning how to do our experiments using Next-Gen
sequencing.
Once I have learned this technology and taught it to my Research group, then I will teach and use this
technology in my Cell Physiology lecture and laboratory course. This course has 48 students in it including
students who are deaf and hard of hearing. RIT has the National Technical Institute for the deaf (NTID) and
students enrolled in NTID often enroll in the College of Science and take our biotechnology courses.
E. I agree to administer the GCAT-SEEK pre- and post-activity assessment test for students and to complete the
faculty post-utilization survey. X yes, ____ no
F. Describe any other broader impact or intellectual merit considerations.
I hope to eventually set up training for other undergraduate colleges in the Upstate NYS area. Several faculty in
undergraduate institutions are VERY excited about learning and using this technology.
References
Authors listed in bold are undergraduate students. The abstracts are available online.
1. H. Amin, C. Alexander, I. M. Evans, “Comparing the Transcriptional Profile of Different [PSI+] and [psi-] Yeast
Strains Grown under Stressful Conditions”, School of Biological Sciences, Rochester Institute of Technology,
Rochester, NY,. Abstract 2402, Presented at the American Society of Cell Biology Meetings held in Philadelphia,
PA, December, 2010.
2. P. Patil1, J. Schulman1, B. A. Dennis2, E. S. Antonio1, I. M. Evans1, Microarray Analysis to Probe Gene
Expression in [PSI+], a Prion-Containing Yeast Cell.
School of Biological Sciences, Rochester Institute of Technology, Rochester, NY, 2Biomedical Sciences, Rochester
Institute of Technology, Rochester, NY. Abstract 503, Presented at the American Society of Cell Biology Meetings
held in Denver, CO, December, 2011.