Browsing by Author "William Thompson, Committee Co-Chair"

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Genomic and Molecular Analyses of the Core DNA Replication Machinery in Plants
(2008-04-04) Shultz, Randall William; Rebecca Boston, Committee Member; Jeffrey Thorne, Committee Member; William Thompson, Committee Co-Chair; George Allen, Committee Co-Chair; Steven Spiker, Committee Member
Accurate and complete DNA replication is essential for maintaining the integrity of the genome. In eukaryotes, this process requires the coordinated action of numerous molecular machines. Based on yeast and animal model systems, we defined a set of fifty-one "core DNA replication proteins" that are integral to the initiation, DNA synthesis, and Okazaki fragment maturation functions of DNA replication. We used computational analyses to identify putative homologs in the genomes of two plants, Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice), providing the first comprehensive view of the core DNA replication machinery in plants. Our results indicated that the overall composition of this apparatus is conserved, but plants are unique in that multiple DNA replication genes exist as small gene families. Fourteen of the genes we annotated in this study have not been previously reported in the literature, and we have provided revised gene models for seventeen plant proteins. To better understand how the DNA replication machinery functions in plants, we cloned multiple subunits of the pre-replication complex (pre-RC) from Arabidopsis and generated antibodies against four key components of this complex — AtORC1, AtORC2, AtMCM5, and AtMCM7. We demonstrated that the pre-RC is developmentally regulated in Arabidopsis and, consistent with a role in DNA replication, is abundant in proliferating tissues. We used immunocytochemical and biochemical methods to characterize MCM7 in plants. We observed two distinct localization patterns for plant MCM7 proteins. In most cells, MCM7 was nuclear and colocalized with DNA. In a small fraction of cells, MCM7 was dispersed throughout the cytoplasmic compartment. Biochemical analysis confirmed that MCM7 binds to chromatin and that it is present in the nucleus at least during the G1, S and G2 cell cycle stages. Together, these analyses support a model where the MCM complex is loaded onto DNA in late M and early G1, released into the nucleoplasm during S phase followed by a brief dispersion into the cytoplasmic compartment concurrent with nuclear envelope breakdown in mitosis.
The Use of Flow Cytometry to Investigate the Effects of Matrix Attachment Regions on Transgene Expression in Plant Cells.
(2005-02-14) Halweg, Christopher Jay; Arthur Weissinger, Committee Member; William Thompson, Committee Co-Chair; Steven Spiker, Committee Co-Chair; Dominique Robertson, Committee Member
Many studies in both plant and animal systems have shown that Matrix Attachment Regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. MARs are AT-rich sequences of DNA that bind in vitro to the proteinacous filament-like structure within the nucleus called the nuclear matrix. In our investigation of transgenic Nicotiana tabacum NT-1 cells in culture, we have observed that transgene expression is often variegated. In other words, some cells in an isogenic population do not express the transgene, and/or other cells within the same population express the transgene at varying levels. The question was raised: Do MARs increase transgene expression by altering variegation? More specifically, do MARs increase the percentage of cells expressing the transgene, increase the magnitude of expression in expressing cells, or both? In order to address these questions, it was necessary to quantitate transgene expression variegation at the resolution of individual cells. We chose to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells by flow cytometry. In order to analyze individual cells and because NT-1 cells in culture grow as filaments, it is necessary to prepare protoplasts that can pass one at a time through the flow cell of the flow cytometer. We found that current flow cytometry methods for measuring GFP expression in plants were susceptible to debris resulting from protoplast preparations. We observed that when the plasma membrane of protoplasts is breached, GFP diffuses out into the medium, and flow cytometric measurements of these non-viable protoplasts imply they do not express GFP. This debris can overestimate the proportion of non-expressing cells in the population. In order to correct this problem, we used an approach called a dye exclusion test. Because propidium iodide enters protoplasts to stain nuclei only when the plasma membrane is breached, debris that stains with this dye can be removed from our analysis. Using this approach we were able to quantitate GFP expression in individual cells without complications from debris. We used flow cytometry to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells from lines transformed by Agrobacterium. We find that in this system the Rb7 MAR increases GFP expression 2-4 fold. This increase is caused by both an increase in the percentage of expressing cells and an increase in the magnitude of expression in expressing cells. Cell lines transformed with MAR-containing vectors averaged 27-39% more cells expressing GFP and these cells expressed GFP at 2-3 fold higher levels than cells transformed with control constructs. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from a population of cell lines obtained by liquid culture of entire Agrobacterium co-cultivation plates. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression. Our results indicate that the Rb7 MAR increases the frequency of transgene expression, presumably by reducing gene silencing, while also increasing the levels of expression in expressing cells, perhaps through an enhancer-like activity.