Date on Master's Thesis/Doctoral Dissertation

5-2014

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Feldhoff, Richard C.

Committee Co-Chair (if applicable)

Feldhoff, Pamela W.

Committee Member

Feldhoff, Pamela W.

Committee Member

Gregg, Ronald G.

Committee Member

Geoghegan, Thomas E.

Committee Member

Perlin, Michael H.

Committee Member

Voss, S. Randal

Subject

Lungless salamanders--Reproduction; Pheromones

Abstract

Pheromones are an important type of chemical cue used by most animals to convey information between individuals. For more than 100 million years, male plethodontid salamanders have utilized a system of non-volatile, proteinaceous pheromones to regulate female mating behavior and receptivity. One of these pheromone components, Plethodontid Modulating Factor (PMF), is a hypervariable protein related to the three-finger protein (TFP) superfamily. Previous studies revealed that PMF persists as a rapidly evolving multi-isoform mixture. However, many characteristics of PMF as a pheromone remained undetermined, including gene structure and transcriptional regulation, translational regulation, protein structure, evolutionary mechanisms, and the isoform effects on female behavior and neurophysiology. Therefore, the broad aim of this dissertation was to characterize the mechanisms of action and evolution for PMF using the red-legged salamander, Plethodon shermani. The molecular and proteomic diversity of PMF was determined by RT-PCR and mass spectroscopy. The PMF complex is the product of at least 13 gene duplications in 3 gene classes containing highly conserved 5’ and 3’ untranslated regions (UTRs). These UTRs are bound by cold inducible RNA binding protein, which likely plays a key role in coordinating expression of the many diverse PMF isoforms during gland development. Using mass spectroscopy and multidimensional NMR, the 3D structure of the most abundant PMF isoform was determined to have a novel structure compared to all other TFPs. In particular, an altered disulfide bonding pattern promoted greater backbone flexibility in the most rapidly evolving segments of PMF to possibly enhance male pheromone and female receptor interactions. Functional assays testing different mixtures of PMF isoforms revealed that isoform diversity is a key requirement for increasing female receptivity, likely through synergistic interactions in the vomeronasal organ and/or the brain. Examination of pheromones in a different plethodontid species (P. cinereus) revealed that the majority of PMF duplications occurred within the last ~20 million years. In summary, in response to female sexual selection, the PMF gene complex has evolved through an unusual disjunctive evolutionary process as part of a birth-and-death model of gene evolution to permit coordinated expression of dozens of flexible proteins that synergistically function to regulate female behavior.

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