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Richard J. Giannone BS: Animal Science, Minor: Nutrition, Rutgers University, Cook College Oak Ridge National Laboratory |
Description of Research
Mentors: Yie Liu & Hayes McDonald
The elucidation of protein complexes involved in mammalian telomere biology using using tandem affinity purification and 2D liquid chromatography-mass spectrometry. In addition to identifying novel interacting partners of several telomere associated bait proteins (hTRF1, hTRF2, hPOT1, hPINX, hTERT, and mTERT), we also aim to investigate how changes in their phosphorylation states, prompted by exposure to various stressors, correspond to alterations in the makeup of their interacting partners.
Rotation Summary
Mentor: Naima Moustaid-Moussa - Fall 2003
Diet-induced differential gene expression in C57/BL6 mice.
Three groups of mice each fed a different diet (high fat, low fat, & high fat / energy restricted) were compared to one another by various methods. Experimental procedures utilized included physical measurements (body weight, fat pad weight, blood glucose levels), hormonal assays (insulin, leptin, adiponectin), and differential genetic analysis using cDNA microarrays. We found that mice on energy restricted diets lost the most weight, accumulated less fat, and were less insulin resistant than their counterparts. Also, we show that previous studies pointing to the benefits of caloric restriction (aging / cancer resistance) are in accord with our results.
Mentor: Greg Hurst - Spring 2004
Examining microspray ionization mass spectrometry: The robustness of electrospray with the sensitivity of nanospray.
The use of the nanospray ionization (NSI) mass spectrometry is well established and has many advantages over traditional electrospray ionization (ESI) which includes but is not limited to enhanced sensitivity and lower flow rates (less sample needed). However, a problem inherent to the technology is the frequent clogging of the emitter due to it's extremely reduced inner diameter and small needle-like glass tip, a problem rarely experienced with electrospray sources. In order to address this issue, a microspray (MSI) source has been developed which touts nanospray like sensitivity and flow rates with the robustness of electrospray. In this rotation, all three ion sources, ESI, NSI and MSI, were tested and compared to see if MSI was truly able to improve upon both ESI's and NSI's flaws. In addition, MSI was used examine the proteome of adipose tissue and its secreted proteins in order to better understand the role of fat tissue as an endocrine organ.
Mentor: Jay Snoddy - Summer 2004
Using Curated Literature to Identify Hypothetical Relationships Between Genes
The GRIF, or Gene Reference into Function, database is a curated set of publications that have been read, analyzed, summarized, and categorized by real people working at NCBI. Human interaction ensures that these categorized references are more trustworthy than if done by a computer’s AI alone. Each gene loci entered into NCBI’s Entrez Gene points to specific references in the GRIF database. Presumably, this means that this particular GRIF / publication contains information about the function of the particular gene to which it is associated. This information can then be used to make assumptions as to whether gene X is associated with gene Y by factors such as that they appear in the same GRIF publication Z number of times, or that they are linked by some other gene, Q (i.e. if gene X and Q share a GRIF while gene Y and Q share a GRIF, then genes X and Y could be functionally related; X = Q and Y = Q, then X = Y). In this project, we choose to use the GRIF publication database for the species Mus musculus in an effort to find these gene-gene associations so that this information can be used to generate hypotheses about the interrelationship of said genes.
Mentor: Yie Liu - Summer 2004
A Shotgun-based Strategy to Identify Novel Telomere-associated Protein Complex Components via Tandem Affinity Purification (TAP) and Mass Spectrometry (MS)
Telomerase or telomere associated proteins are essential for both telomere length regulation and protection. Erosion of the telomere has been linked to aging, premature aging syndromes, and cancer. Therefore, a more complete picture depicting the interplay of telomeric proteins with the telomere will significantly contribute to the understanding of its length regulation and, in turn, aging and cancer. In this study we systematically investigate telomerase and telomere associated protein complexes with the goal of identifying new protein components. To accomplish this task, TRF2, which is involved in telomere length regulation, was cloned into several novel Tandem Affinity Purification (TAP) vectors. The features of the TAP tag allow for a more robust and rapid enrichment of protein complexes from a relatively small number of cells. Enriched protein mixtures are then identified using a shotgun-based MS approach where the purified sample is digested to peptides and separated / analyzed by 2D liquid chromatography (MudPIT: SCX by RP) coupled with tandem MS. Initial work utilizing this approach was successful in identifying numerous interacting proteins, many of which were known to be telomere associated. More importantly, we were able to identify a few novel interactions, one of which has been confirmed to play a role at the telomere.
Publications
- Wang Y, Erdmann N, Giannone RJ, Wu J, Gomez M, Liu Y. 2005. An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres. Proceedings of the National Academy of Sciences. 102(29): 10256-60.
- Wang Y, Giannone RJ, Liu Y. 2005. Telomere Sister Chromatid Exchange in Telomerase Deficient Murine Cells. Cell Cycle. 4(10): 1320-22.
- Zylstra GJ, Cigolini JF, Puga C, Chang HK, Giannone R, and Dennis JJ. 2000. Identification and functional characterization of oxygenases from Mycobacterium strains. Abstr. Soc. Indust. Microbiol., S12, p. 63.