Browsing by Author "Blacklock, Brenda J."
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Item Characterization of a fatty acid elongase condensing enzyme by site-directed mutagenesis and biochemical analysis(2014) Hernandez-Buquer, Selene; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Li, LeiFatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.Item DNA Recognition and Cleavage by Phenyl-Benzimidazole Modified Gly-Gly-His-Derived Metallopeptides(2010-04-08T16:20:15Z) Wang, Tianxiu; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Naumann, Christoph A.Metallopeptides of the general form M(II)∙Gly1-Gly2-His induce DNA strand scission via minor groove interactions. This peptide system can serve as a nucleic acid-targeted cleavage agent – either as an appendage to other DNA binding agents, or as a stand alone complex. In an effort to further our knowledge of DNA recognition and cleavage, a novel series of phenyl-benzimidazole modified Gly-Gly-His-derived metallopeptides was synthesized via solid phase methods and investigated. The new systems allow the formation of additional contacts to the DNA minor groove through the incorporation of a DNA binding phenyl-benzimidazole moiety, thus strengthening the overall binding interaction and further stabilizing the metal complex-DNA association. In addition, how Lys side chains and an amidinium group influence the efficiency of DNA cleavage was also studied. DNA cleavage studies suggested that the phenyl-benzimidazole-modified Gly-Gly-His metallopeptides possess enhanced DNA cleavage abilities. In particular, when amidines are placed on the benzimidazole moieties, these moieties appeared to play an important role in increasing the DNA cleavage activity of the metal complex, most likely through an enhanced electrostatic attraction to the DNA.Item Fatty acid elongation by ELOVL condensing enzymes depends on a histidine nucleophile(Springer, 2021-06) Blacklock, Brenda J.; Chemistry, School of ScienceItem LIPIDOMIC PROFILING OF DICTYOSTELIUM DISCOIDEUM(2012-08-27) Birch, Garrison L.; Minto, Robert; Blacklock, Brenda J.; McLeish, Michael J.The lipid profile of Dictyostelium discoideum, a cellular slime mold found evolutionarily between plants and animals, has never been clearly defined. To address this, the fatty acid content of vegetative cells was analyzed by gas chromatography-mass spectrometry of fatty acid methyl esters and their identities verified with synthesized authentic standards. The synthetic scheme developed to produce the unusual fatty acids found in D. discoideum was engineered to afford the labeling of compounds (2H) for use in feeding studies to elucidate the fatty acid elongation and desaturation pathways present in D. discoideum. After establishing the fatty acid profile and acyl metabolic pathway, an initial understanding the complex lipids present in D. discoideum, chiefly sphingolipids, was sought. Triple quadrupole and quadrupole time-of flight mass spectrometers equipped with electrospray ionization sources were used to identify these complex lipids.Item Molecular and Physiological Responses of Soybean (Glycine max) to Cold and the Stress Hormone Ethylene(2019-05) Robison, Jennifer Dawn; Randall, Stephen K.; Balakrishnan, Lata; Watson, John C.; Blacklock, Brenda J.Abiotic stresses, such as cold, are serious agricultural problems resulting in substantial crop and revenue losses. Soybean (Glycine max) is an important worldwide crop for food, feed, fuel, and other products. Soybean has long been considered to be cold-intolerant and incapable of cold acclimation. In contrast to these reports, this study demonstrates that cold acclimation improved freezing tolerance in the domestic soybean cultivar ‘Williams 82’ with 50% enhancement of freezing tolerance after 5.2 +\- 0.6 days of cold exposure. Decreases in light dependent photosynthetic function and efficiency accompanied cold treatment. These decreases were due to an increase in photon dissipation likely driven by a decrease in plastoquinone (PQ) pool size limiting electron flow from photosystem II (PSII) to photosystem I (PSI). Cold-induced damage to operational photosynthesis began at 25 minutes of cold exposure and maximal photosynthesis was disrupted after 6 to 7 hours of cold exposure. Cold exposure caused severe photodamage leading to the loss of PSII reaction centers and photosynthetic efficiency. Comparisons of eight cultivars of G. max demonstrated a weak correlation between cold acclimation and northern cultivars versus southern cultivars. In the non-domesticated soybean species Glycine soja, the germination rate after cold imbibition was positively correlated with seedling cold acclimation potential. However, the overall cold acclimation potential in G. soja was equal to that of domestic soybean G. max reducing the enthusiasm for the “wild” soybean as an additional source of genetic diversity for cold tolerance. Despite being relatively cold intolerant, the soybean genome possesses homologs of the major cold responsive CBF/DREB1 transcription factors. These genes are cold-induced in soybean in a similar pattern to that of the cold tolerant model plant species Arabidopsis thaliana. In Arabidopsis, EIN3, a major component of the ethylene signaling pathway, is a negative transcriptional regulator of CBF/DREB1. In contrast to AtEIN3 transcript levels which do not change during cold treatment in Arabidopsis, we observed a cold-dependent 3.6 fold increase in GmEIN3 transcript levels in soybean. We hypothesized that this increase could prevent effective CBF/DREB1 cold regulation in soybean. Analysis of our newly developed cold responsive reporter (AtRD29Aprom::GFP/GUS) soybean transgenic lines demonstrated that inhibition of the ethylene pathway via foliar sprays (AVG, 1-MCP, and silver nitrate) resulted in significant cold-induced GUS activity. Transcripts of GmEIN3A;1 increased in response to ethylene pathway stimulation (ACC and ethephon) and decreased in response to ethylene pathway inhibition in the cold. Additionally, in the cold, inhibition of the ethylene pathway resulted in a significant increase in transcripts of GmDREB1A;1 and GmDREB1A;2 and stimulation of the ethylene pathway led to a decrease in GmDREB1A;1 and GmDREB1B;1 transcripts. To assess the physiological effects of these transcriptional changes; electrolyte leakage, lipid oxidation, free proline content, and photosynthesis were examined. Improvement in electrolyte leakage, a measure of freezing tolerance, was seen only under silver nitrate treatment. Only 1-MCP treatment resulted in significantly decreased lipid oxidation. Transcripts for CBF/DREB1 downstream targets (containing the consensus CRT/DRE motifs) significantly decreased in plants treated with ethylene pathway stimulators in the cold; however, ethylene pathway inhibition generally produced no increase over basal cold levels. To identify if GmEIN3A;1 was capable of binding to GmDREB1 promoters, the negative regulator GmEIN3A;1 and the positive regulator GmICE1A were cloned and expressed in Escherichia coli (E. coli). Preliminary binding results indicated that GmEIN3A;1 can bind to a double stranded section of the GmDREB1A;1 promoter containing putative EIN3 and ICE1 binding sites. GmICE1A is capable of binding to the same section of the GmDREB1A;1 promoter, though only when single stranded. Additional experiments will be required to demonstrate that GmEIN3A;1 and GmICE1A are capable of binding to the GmDREB1A;1 promoter and this work provides the tools to answer these questions. Overall, this work provides evidence that the ethylene pathway transcriptionally inhibits the CBF/DREB1 pathway in soybean through the action of GmEIN3A;1. Yet when GmCBF/DREB1 transcripts are upregulated by ethylene pathway inhibition, no consistent change in downstream targets was observed. These data indicate that the limitation in cold tolerance in soybean is due to a yet unidentified target downstream of CBF/DREB1 transcription.Item Monitoring, characterizing, and preventing microbial degradation of ignitable liquids on soil(2013) Turner, Dee Ann; Goodpaster, John V. (John Vincent); Michalski, Greg M.; Blacklock, Brenda J.; Siegel, Jay A.; Long, Eric C. (Eric Charles)Organic-rich substrates such as soil provide an excellent carbon source for bacteria. However, hydrocarbons such as those found in various ignitable liquids can also serve as a source of carbon to support bacterial growth. This is problematic for fire debris analysis as samples may be stored at room temperature for extended periods before they are analyzed due to case backlog. As a result, selective loss of key components due to bacterial metabolism can make identifying and classifying ignitable liquid residues by their chemical composition and boiling point range very difficult. The ultimate goal of this project is to preserve ignitable liquid residues against microbial degradation as efficiently and quickly as possible. Field and laboratory studies were conducted to monitor microbial degradation of gasoline and other ignitable liquids in soil samples. In addition to monitoring degradation in potting soil, as a worst case scenario, the effect of soil type and season were also studied. The effect of microbial action was also compared to the effect of weathering by evaporation (under nitrogen in the laboratory and by the passive headspace analysis of the glass fragments from the incendiary devices in the field studies). All studies showed that microbial degradation resulted in the significant loss of n-alkanes and lesser substituted alkylbenzenes predominantly and quickly, while more highly substituted alkanes and aromatics were not significantly affected. Additionally, the residential soil during the fall season showed the most significant loss of these compounds over the course of 30 days. To combat this problem, a chemical solution is to be immediately applied to the samples as they are collected. Various household and commercial products were tested for their efficacy at low concentrations to eliminate all living bacteria in the soil. Triclosan (2% (w/v) in NaOH) proved to be the most effective at preserving ignitable liquid residues for at least 30 days.