Date of Award

2009

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Carolyn Bentivegna

Committee Member

Yufeng Wei

Committee Member

Jane L. Ko

Keywords

GPCR characterization, Halobacterium salinartum (H sal), Bacterioopsin gene (Bop), Stem loop structure

Abstract

In pursuit of GPCR characterization, our lab chose to work with a model organism Halobacterium salinarum (H sal). This organism contains the bacterioopsin gene (bop), which encodes the protein precursor of the purple membrane protein bacterioopsin (Bop). The bacterioopsin gene (bop) was chosen as the reporter for the study. The putative stem-loop structure within the bop gene is considered to be a key component of the molecular machinery that regulates Bop synthesis. Previous work in our lab has indicated that single base pair disruptions dramatically reduced stem-loop stability (differential scanning calorimetry (DSC), circular dichroism (CD) and ultraviolet (UV) spectroscopy analysis). The work presented here evaluated the influence of cooperative base pairing on stem-loop stabilization (Mfold algorithm predictions) and the relationship between stem-loop stability and bR production in-vivo. The approach involved subcloning mutant oligomers into a vector designated as pENDS and subsequently cloning the product into an expression vector designated as pHex. The pHex vector was then expressed in Il.sal. In order to check the validity of the cloning, the vector was transformed back into E.coli and the plasmid DNA was extracted and sequenced.Results of this preliminary mutational study of the 5' bop gene stem-loop structure indicated that the stem-loop is indeed present in-vivo in Hsal. Further the study mdicated that an increase in the stability of the stem-loop decreases the production ofbR significantly, and the decrease in the stability of the stem-loop also decreases the production of bR. The decrease in stability was not directly proportional to the increase in predicted 6-G value. However, the increase in stability was directly proportional to the decrease in the predicted 6-G value. This work has found a correlation between the stem- loop structural stability and bR accumulation, thereby demonstrating that molecular machinery responsible for the production of bR in Hsal is highly optimized. More quantitative methods like mRNA analysis can be used to further establish these findings.

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