DONDE ANDERSON

RESEARCH RATIONALE	Understanding the function of phytochromes as dictated by protein conformation allows researchers to identify the mechanisms that regulate plant activity. This activity can be influenced by specifically altering plant genotypes through recombinant DNA processes. These genetically altered organisms will have the capability of surviving in climates that would usually destroy native organisms and have agriculture and forestry value. Donde and Dr. Song are working to identify the relationship between the conformations of phytochrome and plant activities, ultimately, this research will lead to the production of new and vital plants.




RESEARCH BACKGROUND	The Phytochrome family of plant proteins monitors the light environment and dictates patterns of gene expression that enable the plant to optimize growth and development according to environmental conditions. The activities of germination, growth, flowering and reproduction are dependent upon the action of these proteins which in turn is dictated by specific conformations expressed in the proteins.
	The phytochrome protein exists as two spectrally distinct forms; Pr, the red light absorbing form (l_max = 660 nm) and the Pfr, far-red light-absorbing form (l_max = 730 nm). These two conformational forms are inter-convertible and regulated by the wavelength of light. It is believed that Pfr form is the active form and the Pr is the inactive form.
	For a more thorough explanation of phytochrome and its role in photomorphogenesis, click here.

RESEARCH PROCEDURE	Donde, with the assistance of predoctorate student,Jeong-Il Kim, will create several DNA mutants using deletion and site-directed mutagenesis, express the mutant proteins in bacterial and yeast cells and then characterize them using various techniques.

	The initial phase of Donde's project requires that they obtain mutated DNA. This mutation process is performed by two separate processes, deletion and site-directed mutagenesis. Deletion is the process of excising certain segments of DNA from the gene that codes for phytochrome. The expressed phytochrome will then have segments of amino acids missing. The second process Donde will perform is site-directed mutagenesis. This process includes site-specific changes in DNA nucleotides which substitute single residues in the protein. The lab is focused on changing cysteine residues for serine residues. The mutations then have to be amplified, verified and purified using different vectors and lab processes.

	To get an overview of the process that is involved in DNA cloning, click here.

	Link to Donde's research in creating mutated DNA plasmids, isolation and purification procedures by clicking on the "Subcloning" Leaf.



	Protein Expression involves the ligation of the recombinant plasmid into an expression vector and then transforming the vector/plasmid into a host cell where the host will produce a protein from the recombinant DNA. The intent is to be able to identify functional changes in the phytochrome which will allow Donde and Jeong-Il to identify the structure-function relationship of phytochrome.
	The host cells that Donde will be using are Escherichia coli and Pichia pastoris (yeast). The E. coli is a prokaryote while the yeast is an eukaryote. For an explanation of why E. coli and yeast are used as host cells, click here.

	Link to Donde's research in phytochrome protein expression by clicking on the "Protein Expression" Leaf.


	Structure-Function relationships between the mutated phytochrome and cell photomorphogenesis will be accomplished by several experiments. Unfortunately this will not be performed by Donde this summer, hopefully she will get a chance to come back to UNL and work on this project.

	DONDE ANDERSON: Structure & Function Relationships of Plant Phytochromes

	Objective: Identify the molecular mechanism based on the two conformations of Plant Phytochromes through mutagenesis and protein characterization

	Laboratory: Dr. Pill-Soon Song