Title

Telomerase gene expression in adult sockeye salmon

Abstract

Research for the following capstone project is to be performed by Jason G. Tayag, under the guidance of Professor Steven Roberts and Graduate Student Caroline Storer. The project is an assay measuring telomerase gene expression in adult sockeye salmon. The results of the study can hopefully help to explain how quickly or how slowly telomeres are shortening in adult sockeye salmon.

Intro

Accurate and complete DNA replication is important for cell division. Mutations in genetic information can result in deleterious phenotypic changes in an organism (Geserick and Blasco 2006). Cells utilize a number of ways to protect against deletions and changes to DNA. One such way to protect against deletion of genetic information is with telomeres. Telomeres are 6-base-pair repeat sequences at the ends of chromosomes that haven’t been known to code for anything Geserick and Blasco 2006). During DNA replication, DNA is packaged into chromosomes. When chromosomes replicate, genetic material on the ends of the chromosomes is deleted (Geserick and Blasco 2006). Telomeres protect genomic DNA from being deleted. Over time, however, telomeres shorten with every cell division – cells divide many times throughout an organism’s life. Telomere shortening may ultimately have links to aging and senescence (Geserick and Blasco 2006).

Salmon are a very interesting organism we can use to study senescence because salmon health declines rapidly after releasing gametes. Studies have been done looking at post- and pre-spawning adults arriving at their natal stream, as well as juvenile telomere lengths. I want to focus on telomere lengths of adults in the ocean that may be getting ready to return to their natal stream to spawn. My hypothesis is that salmon at sea undergo rapid telomere shortening because that is where growth is occurring most rapidly. Before they even reach their natal stream, it is likely that telomere lengths have already shortened past critical length. This means that sockeye would already be going through a decline in health before they reach their natal stream. Though looking at actual telomere lengths can be difficult, there is a proximate way to measure telomeres by looking at the expression of the telomerase gene. Telomerase is an enzyme that restores telomeres. High telomerase activity allows us to know that at one particular point in time, telomerase is rapidly restoring telomeres – likely because telomeres are rapidly shortening.

Questions

At what life stage/stages does telomere shortening occur most rapidly?

Is telomerase gene expression high in adult sockeye salmon in the ocean?

Methods

Collection

-Find a salmon fishery where we can obtain flash-frozen adult sockeye salmon tissue samples from 8-10 individuals.

-Research and design primers that will target telomerase gene sequence.

-Extract RNA from tissues, reverse-transcribe RNA to DNA.

-Measure telomerase gene expression by qPCR.

-qPCR a normalizing gene so that we have a standard or a baseline to compare telomerase.

Materials

TRI Reagent, Chloroform, Isopropanol, 75% Ethanol, DNA Free DNase kit, 0.1% DEPC-H2O, Oligo dT, Nuclease-free water, M-MLV 5X Reaction Buffer, dNTPs, M-MLV RT, GoTaq Master Mix Forward and Reverse Primers (need to be designed), Pipets, pipet tips, Microcentrifuge tubes, qPCR tubes, Thermocycler, -80°C freezer, -20° freezer, centrifuge, microcentrifuge, plastic plungers, spectrophotometer.

Data Organization

-Transform C(t) values into relative gene expression values.

Timeline, Products

At the end of my research, my goal is to be able to write a scientific paper on my findings.

-January 20, 2011 (2:00 PM): Next meeting with Professor Roberts.

-Before January 20: try to come up with primer design for sockeye salmon telomerase gene.

-The limiting step is finding adult sockeye salmon samples and getting them frozen and shipped. My goal is to obtain all of my samples by February 4, 2011.

-qPCR would take 2-3 weeks to complete and analyze. I need to design and order primers, which takes a few days. Most of the reagents for qPCR are easy to obtain.

-End of quarter: qPCR should be complete (data collection complete)

Approval Signatures

We have read and discussed the above proposal thoroughly and we believe this is an achievable yet challenging project for the student named. We have also discussed how the student will get any needed supplies, etc. for this project.

                                                                              1/14/2011        

(Faculty Sponsor’s Signature)                                   ______________________  

Date

 Steven Roberts

______________________________________

(Printed name of Faculty Sponsor)