Date

2005

Document Type

Master's Thesis

Degree Name

Master of Science (M.S.)

Department

Moss Landing Marine Laboratories

Abstract

Although the number of fluorescent protein (FP) genes cloned from the GFP family continues to increase, few studies of GFP-type pigments in non-bioluminescent, non-symbiotic organisms have been attempted. The first goal of this study was to locate, clone, characterize, and analyze fluorescent proteins from an organism exhibiting these traits in order to better understand their evolution and function. I successfully cloned two full-length GFP homologs by applying a FACS-based screening method to a cDNA library constructed from a temperate corallimorpharian, Corynactis californica. The full-length coding regions of each gene were subcloned into an expression vector and bacterial cultures were used to express the proteins. Spectral properties of purified proteins were characterized and chromophore maturation behavior was examined. Phylogenetic methods were also used to analyze the new gene sequences in relation to homologous GFP family members. After discovering two GFP-like proteins in a single red morph, I investigated six additional morphs of Corynactis californica, and found indications of a variety of fluorescent pigments based on fluorescence emission spectra from live specimens. The second goal of this study was to identify and describe the variation in fluorescent pigments among morphs of C. californica and to relate the in vivo emission patterns and colors to FP genes cloned from and expressed in each morph. Specifically, I found that all morphs express a similar suite of GFP-like proteins, generated by at least three to four genetic loci, which code for three colors: green, orange, and red. The genes exhibit tissue-specific expression patterns that differ by morph, and two major expression patterns emerged. Sequence and phylogenetic analyses comparing the new FP genes from C. californica to one another and to homologous members of the GFP family indicate that FP genes from this species are most closely related to one another, but that FP genes arose in an ancestor to the Anthozoa before speciation events separating anthozoan subclasses, including the Corallimorpharia. Possible ecological roles of variations in fluorescent pigmentation among morphs of C. californica are also discussed.

Comments

Thesis (M.S.) Division of Science & Environmental Policy. Moss Landing Marine Laboratories

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