Title page for ETD etd-04282011-152449


Type of Document Master's Thesis
Author Chiragh, Imran
URN etd-04282011-152449
Title Devloping a Tool to Characterize the Ultradian Rhythm in Diploid Saccharomyces cerevisiae Using the Reporter Gene Green Fluorescent Protein
Degree Master of Science in Chemical Engineering (M.S.Ch.E.)
Department Chemical Engineering
Advisory Committee
Advisor Name Title
Benton, Michael Committee Chair
Henry, James Committee Member
Hjortsø, Martin Committee Member
Keywords
  • ultradian rhythm
  • saccharomyces cerevisiae
  • reporter gene
  • green fluorescent protein
  • yeast
  • budding yeast
  • circadian rhythm
  • biological rhythm
Date of Defense 2011-04-25
Availability unrestricted
Abstract
Biological rhythms control many temporal behaviors of organism, such as the sleep cycle, hearts rhythms, seasonal animal migrations etc. Understanding these rhythms would provide insight into the temporal process of living organisms. Saccharomyces cerevisiae, a budding yeast, is an ideal model organism to study biological rhythms in eukaryotic cells because of its sequenced genome and discerned processes. By characterizing the biological rhythm in budding yeast, insight can be gained into more complex organisms.

Previous studies have exhibited oscillatory behavior of oxygen consumption and determined that deletion of the GTS1 gene dissipates this rhythm. However, to further understand the specific behavior of this gene, GTS1 needs to be simultaneous monitored as it is expressed. In this study to monitor this ultradian rhythm regulating gene, a promoter-reporter construct was inserted through homologous recombination to track the expression of GTS1 in a diploid yeast strain, BY 4743. The promoter-reporter construct replaced one copy of the GTS1. As the GTS1 was expressed, the construct was expressed and detected by its reporter gene, green fluorescent protein (GFP). Synchronization of the cell cycle and ultradian rhythm was achieved by addition of hydroxyurea and nocodazole to the growth media. GFP levels were quantified by flow cytometry, with samples taken every 10 minutes.

The results showed GFP expression level from the transformed yeast strain exhibiting a 3.33-fold increase relative to the non-transformed yeast strain. GFP expression yielded a biological rhythm with two identifiable periods, each with a 70 minute period. The first oscillation began at time zero and had a GFP expression maximum of 2.96 times the control level and a minimum of 2.62. The second oscillation began at 70 minutes had a GFP expression maximum of 3.09 times the control and a minimum of 2.76. The biological rhythm observed was shorter than its own cell cycle, roughly 111 minutes. Oscillatory behavior was observed as long as the culture remained synchronous.

This study characterized the behavior of GTS1, an ultradian rhythm gene. By characterizing the behavior of this gene in S. cerevisiae, homologous genes in more complex organisms such as rodents or humans can be better understood. By extrapolating temporal behavior in yeast to humans, a cost effective drug prescreening can be implemented to evaluate possible biological rhythmic changes.

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