2006 Summer Undergraduate Research and Creative Achievements Forum (MU)
https://hdl.handle.net/10355/548
The items in this collection are the scholarly output of MU students conducting research through the summer.2024-03-28T16:56:29ZAdventures in Arabidopsis: Two projects investigating iron homeostasis in plants [abstract]
https://hdl.handle.net/10355/613
Adventures in Arabidopsis: Two projects investigating iron homeostasis in plants [abstract]
Cooper, Charles B.; Charlson, Dirk V.; Maurer, Alberto, 1970-; Rogers, Elizabeth E.
In order to identify genes involved in iron homeostasis, a novel genetic screen was developed. Seed from a line transgenic for Green Fluorescent Protein (GFP) driven by the Fer1 promoter, a gene whose mRNA expression levels are increased during iron sufficiency and thus reflects iron status in plants were mutagenized with EMS and screened for high or low GFP fluorescence relative to non-mutagenized transgenic controls. From this screen, approximately 300 putative mutants for iron homeostasis were identified based on either high or low fluorescence relative to the transgenic control on iron-sufficient media. In order to more accurately determine the GFP protein level in the plants, total protein from each line grown on iron-sufficient media for 14 days then transferred to iron-deficient or fresh iron-sufficient media is being extracted, and will be separated by SDS-PAGE, and probed for GFP using a two-antibody detection system. One mutant, OAF102, had been previously identified for its constitutive fluorescence during both iron-sufficient and -deficient conditions three days after transfer, and had been shown to have uncoupled GFP and Fer1 mRNA levels between iron-sufficient and -deficient media at this point. OAF 102 and the non-mutagenized transgenic control were grown for 14 days on iron-sufficient media, then transferred to iron-sufficient or -deficient media. GFP and Fer1 mRNA levels were determined by northern blotting at 2, 3, 4, and 5 days after transfer. Two repetitions of the experiment were performed. Neither one showed the uncoupling seen in previous experiments at three days after transfer. OAF 102 may have constitutively elevated GFP mRNA, but normal regulation of Fer1 mRNA levels. Results have not been consistent and more experiments are being done. Four closely related transcription factors, bHLH's 038, 039, 100, and 101, had previously been shown to be upregulated in Arabidopsis during iron deficiency, but T-DNA insertion lines for each transcription factor did not show any changes in ferric-chelate reductase activity, chlorophyll content, or iron content, all of which are indicators or iron status in the plant. In order to further evaluate the potential role of these transcription factors in iron homeostasis, crosses were performed to create five double mutants with T-DNA insertions in two of
Abstract only available; Faculty Mentor: Elizabeth Rogers, Biochemistry
2006-01-01T00:00:00ZThe affects of genistein on the Bradyrhizobium japonicum bacterial transcriptome [abstract]
https://hdl.handle.net/10355/920
The affects of genistein on the Bradyrhizobium japonicum bacterial transcriptome [abstract]
Ward, Stefanie; Franck, William L., 1980-; Chang, Woo-Suk; Stacey, Gary, 1951-
Bradyrhizobium japonicum forms a symbiotic relationship with the roots of the soybean plant (Glycine max). This bacterium is of great importance because of its ability to provide the soybean with a source of nitrogen by the conversion of atmospheric dinitrogen to ammonia. In order to establish this symbiosis, the bacteria must attach to the root hair surface and initiate development of a root nodule. The bacterium responds to plant flavonoids through production of nod factor, a product of the nod genes, which facilitates bacterial root entry and initiates nodule organogenesis. Therefore, we have investigated the expression of nod genes after treatment with a plant derived flavonoid inducer, genistein. Free-living cultures of Bradyrhizobium japonicum were treated and cells were harvested six hours after treatment and total RNA was extracted. Semi-quantitative and quantitative RT-PCR was performed to confirm induction of the nodY and nodC genes. The RT-PCR results confirmed that nodY and nodC are upregulated in the presence of genistein compared to an ethanol-treated control. To further define genistein regulation on the nod genes, a DNA microarray experiment was performed on the above extracted RNA to define the bacterial transcriptional response to genistein, the results of which will be presented.; Bradyrhizobium japonicum forms a symbiotic relationship with the roots of the soybean plant (Glycine max). This bacterium is of great importance because of its ability to provide the soybean with a source of nitrogen by the conversion of atmospheric dinitrogen to ammonia. In order to establish this symbiosis, the bacteria must attach to the root hair surface and initiate development of a root nodule. The bacterium responds to plant flavonoids through production of nod factor, a product of the nod genes, which facilitates bacterial root entry and initiates nodule organogenesis. Therefore, we have investigated the expression of nod genes after treatment with a plant derived flavonoid inducer, genistein. Free-living cultures of Bradyrhizobium japonicum were treated and cells were harvested six hours after treatment and total RNA was extracted. Semi-quantitative and quantitative RT-PCR was performed to confirm induction of the nodY and nodC genes. The RT-PCR results confirmed that nodY and nodC are upregulated in the presence of genistein compared to an ethanol-treated control. To further define genistein regulation on the nod genes, a DNA microarray experiment was performed on the above extracted RNA to define the bacterial transcriptional response to genistein, the results of which will be presented.
Abstract only available; Faculty Mentor: Gary Stacey, Plant Microbiology and Pathology
2006-01-01T00:00:00ZAnalysis of HIV-1 reverse transcriptase inhibition by multiple RNA aptamers
https://hdl.handle.net/10355/615
Analysis of HIV-1 reverse transcriptase inhibition by multiple RNA aptamers
Cutler, Robin; Rose, Ferrill Franklin, 1978-; Held, Daniel; Burke, Donald H.
Rapidly increasing resistance to currently available HIV-1 drugs has prompted the exploration of therapies less susceptible to resistance development. Anti-HIV-1 treatments often target reverse transcriptase (RT), the viral protein that polymerizes the integrated DNA copy of the viral RNA genome. For many such drugs, resistance requires only a few amino acid changes in the RT protein. Aptamers are small, single stranded nucleic acids that form unique three-dimensional structures allowing specific binding to molecular targets. When bound to proteins, aptamers often hinder their native function. RNA and DNA aptamers that bind to viral proteins and inhibit HIV-1 at several stages of infection have been isolated. Burke et al, (1996) have proposed that variability in anti-RT aptamer structures may overcome the development of resistance because of different contacts with the RT protein. Here, multiple anti-HIV-1 RT RNA aptamers (118 or 134 nucleotides in length) were screened for inhibition of RT-catalyzed DNA polymerization. Aptamer inhibition of RT activity was measured using an HIV-derived synthetic template and fluorescently-labeled primers, and by quantifying the amount of fully extended primer. Sixty-two aptamers were screened and grouped according to inhibition performance. In the “BEST” category, twenty-two aptamers displayed ~100 percent inhibition at the highest aptamer concentration (100 nM) and half-maximal inhibition (IC50) values of less than 3 nM. The “VERY GOOD” category contains twenty aptamers with greater than 90 percent inhibition at the highest concentration and IC50 values between 3 nM and 9.5 nM. Of the remaining samples, “MODEST” aptamers yielded 25 to 80 percent inhibition and “POOR” aptamers showed less than 20 percent inhibition at the highest aptamer concentration. Furthermore, sequence and structural analyses may reveal variations in the aptamer-protein interaction between potent aptamers. These data may define the critical interactions between the aptamer and RT, increasing inhibition potency while reducing susceptibility to resistance.
Abstract only available; Faculty Mentor: Donald H. Burke, Molecular Microbiology and Immunology
2006-01-01T00:00:00ZAnalysis of maize mitochondrial genome structures in vivo [abstract]
https://hdl.handle.net/10355/623
Analysis of maize mitochondrial genome structures in vivo [abstract]
Doyle, Anne; Westgate, Leah; Wolf, Mark; Newton, Kathleen J.
Mitochondria are organelles containing their own genomic DNA that are found in most eukaryotic cells. In contrast to those of animals, plant mitochondrial genomes are large and very complex in structure. While maize mitochondrial DNA can be mapped as a large single circle, it has not been observed as such. Thus, the in vivo organization of the mitochondrial genome is not known. Using the B37 maize inbred line, which has a 570 kilobase (kb) mitochondrial genome, observations were made regarding the possibility of sub-genomes resulting from recombination between repeats. Mitochondria were isolated from fresh B37 seedling shoots and were placed on slides to be observed using the FISH (Fluorescent in situ Hybridization) technique. Two probes were used, cosmid 6 in Alexafluor 488 (green) and cosmid 19 in Texas Red (red). These cosmids are separated in the mitochondrial genome by approximately 350kb. They also occur on separate sides of a small repeat that is known to recombine frequently. If the genome is intact the two colors should overlap and produce a yellow color. If there are sub-genomes, red and green should be seen individually. The mitochondria were observed on an Olympus Spectral Imaging Microscope and the genome appeared to be fragmented most of the time. Five slides were counted and 31,077 spots were observed. The red: green: yellow ratio was 6.44 : 2.96 :1.00. Stretching out of an intact mitochondrial genome could cause apparent sub-genomes. However, it is more likely that there are sub-genomes present in separate mitochondria because small mitochondria (about one micron) were selected during the isolation procedure. This is small enough that the microscope should have seen the signal as overlapping. Future studies could extend the analysis into other maize lines and to other types of plants to determine if the sub-genomic structures are universal.
Abstract only available; Faculty Mentor: Dr. Kathleen Newton, Biological Sciences
2006-01-01T00:00:00Z