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Date of Award

Spring 1997

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biology

First Advisor

Dr. Brad Goodner

Abstract

Growth and development is a complex process that must be carefully and specifically regulated by individual cells and my multicellular organisms. As growth occurs, directional axes are established both within single cells and within the organism as a whole. This issue is important to all organisms which have polarized structures and processes, and is therefore an area of great interest to biologists.

Because all organisms set up directional axes during development, the issue of a conserved mechanism for this establishment arises (Drubin, 1991). Animals such as humans have three organismal axes: anterior-posterior, dorsal-ventral, and right-left. Single cells, such as the epithelial cells that line our intestine, and root hairs in plants, within a larger organisms also grow in a polarized way. Are these processes integrated by cells by way of a similar mechanism?

There is some data that supports the hypothesis of conserved cell polarity mechanisms. The actin cytoskeleton is involved in the organization and transport of materials to a particular site in a variety of cells, such as budding yeast, plant root hairs, fungal filaments, and nematode eggs. Proteins which regulate the assembly of the actin cytoskeleton may also be conserved. One such protein is the small GTP-binding protein CDC42p. It shows homology to proteins in the ras superfamily, and more specifically the rho subfamilywithin ras (Chant et al, 1991). It is known to be involved in the cell polarity, cell morphology, localization of secretions, and deposition of cell-surface material (Ziman et al, 1991).

CDC42p and its homologs have been found in all eukaryotic cells, but has been most closely studied in the budding yeast Saccharomyces cerevisiae where it is essential to the assembly of the site for the emergence of a new bud in the cell division process. S. cerevisiae cells mutant in CDC42p try unsuccesfully to bud and instead simply grow larger all over as their actin cytoskeltons are not established (Chant et al, 1991). These mutants also show other abberrent actin containing structures (Ziman et al, 1991). Other proteins are also known to interact with CDC42p in the organization of the actin cytoskeleton and the organization of the bud site itself (figure 1).

This study attempts to elucidate the role that CDC42p plays in the plant Arabidopsis thaliana .. CDC42p is known to exist in plants, but its mode of action is not known. Does it use the same mechanisms and pathways and does it interact with the same types of proteins as in other eukaryotes, or is there some other mechanism at work? To address this question, a powerful and specific method called the two-hybrid screen was used to search an expression A. thaliana library for proteins that would interact with CDC42p from the budding yeast S. cerevisiae. Once isolated, a portion of these proteins were tested to determine the strength of the interaction with CDC42p, and the strongest interactors were sequenced and characterized.

The two-hybrid system used was created by transforming a series of plasmids into the budding yeast S. cerevisiae (Gyuris et al, 1993) The plasmids encode different hybrid proteins and the reporter gene that allows the interaction between CDC42p and the particular protein to be seen. One plasmid encodes a bait protein that is a combination of CDC42p and lex a, a DNAbinding domain. A second encodes other hybrid proteins (gift from Pringle Lab, UNC). These proteins include a library protein from A. thaliana (gift from Goodman Lab, Harvard U.)and a transcription activation domain that will tum on the transcription of the two reporter genes in the system. Each yeast cell will contain a different hybrid protein, and each will be different as it will have a different protein from A. thaliana included in it. The third plasmid contains one of the reporter genes fused to a lex a binding site.

When this system is completed, the proteins will be allowed to interact within the yeast cells (figure 2). The CDC42p hybrid protein will bind to the lex a binding site on the reporter plasmid. If the specific plant protein in the second hybrid protein interacts with the CDC42p bait protein, it will bring the transcription activation domain that is fused to it into close proximity with the reporter gene, and it will cause the transcription of the reporter gene. The reporter gene is a lacZ gene that encodes B-galactosidase, which turns blue in the presence of Xgal. The cells are plated onto media containing Xgal, so any colonies that appear blue are colonies that have in them library proteins that interact with CDC42p.

There is a second reporter that is part of the EGY48 chromosome that is used as a secondary assay to ensure that the positive Xgal assay is a result of actual interaction and not a result of some mutuation in the lacZ gene that turns it on without requiring interaction between the library protein and the CDC42p bait. The same lex a site is present in the EGY 48 chromosome just before a gene whose protein product is needed for the cell to make its own leucine. Some of the selective media used to grow the cells is free of leucine, so the only cells that can grow must have the interaction in order to make leucine, which is essential for cell growth. Cells that would grow without leucine present (implying that the interaction of the two hybrid proteins turned on the leucine reporter) but without the presence of the second hybrid protein that is only present when cells are grown on galactose/raffinose do not contain indicators, but have a false positive reaction.

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