Whole Genome Analysis Reveals That Conservation and Duplication Gave Rise to the Current V2R Gene Repertoire in Mammals
Alexander Berry, 2012 (Advisor: Dr. Kristen Webb)
The vomeronasal organ (VNO) is the part of the olfactory system that detects specific semiochemicals known as pheromones, which allow for communication between members of a species, and also affect social and reproductive behaviors. The chemodetection function of the VNO in mammals is facilitated by G protein-coupled receptors (GPCRs) encoded by members of two gene superfamilies: vomeronasal 1 receptor (V1R) and vomeronasal 2 receptor (V2R). Previously, members of the V2R gene family from eight mammalian genomes had been identified and their evolutionary relationships reconstructed. The objective of this study was to use bioinformatics techniques to survey four additional mammalian genomes from the African savannah elephant (Loxodonta africana), the European rabbit (Oryctolagus cuniculus), the guinea pig (Cavia porcellus), and the domestic horse (Equus caballus) for V2R genes and to include these genes in evolutionary analyses. These four mammals were chosen to diversify the group of mammals surveyed for V2R genes. Ninety and fifty-two intact genes were identified in O. cuniculus and C. porcellus, respectively, and these genes appear to have mostly evolved through duplications after the rodent lineages diverged. Two and five intact genes were found in E. caballus and L. africana, respectively, and appear to be conserved, as they are also present in several other species.
Thermodynamic Investigation of Group III Single Nucleotide Bulge Loops and Group II Single Nucleotide Bulge Loops with Adjacent GU Wobble Base Pairs in RNA Duplexes
Anthony Blaszczyk, 2012 (Advisor: Dr. Marty Serra)
Four Group III and seven Group II RNA duplexes containing a single nucleotide bulge loop adjacent to a GU wobble base pair were optically melted, and the thermodynamic parameters (ΔG°37, ΔH°, ΔS°, and TM) were determined for each sequence. Data from this study were combined with data from previous thermodynamic investigations to compare the stability of Group II and Group III bulge loops with an adjacent GU wobble base pair (McMichael, unpublished; Serra, unpublished). The current model to predict the thermodynamic influence of a bulge loop does not consider Group II or Group III bulges with an adjacent GU wobble base pair. In this study Group II and Group III sequences with bulges adjacent to a GU wobble were analyzed and compared to previously studied Group I bulges with an adjacent GU wobble base pair. The identity of the bulge is ambiguous in both Group II and Group III sequences, but the nucleotides that could be bulged were shown to have no influence on the stability of the duplex. Group III bulges were the only group that saw a thermodynamically favorable influence by adding an adjacent GU wobble base pair in place of a Watson-Crick base pair. Group II bulges were shown to only be thermodynamically favorable when the sequence had the potential to form a tandem GU motif. For both Group II and Group III bulges, the potential to form a tandem GU motif was seen to be more important than both the position and orientation of the neighboring GU base pair.
Sequence Comparison and Analysis of the Haemophiulus ducreyi homologue to Escherichia coli tolB
Colleen Dailey, 2012 (Advisor: Dr. Tricia Humphreys)
Haemophilus ducreyi is a gram-negative bacterium that causes the sexually transmitted disease chancroid. Despite several attempts, a vaccine for H. ducreyi has not been developed. Because the bacteria are resistant to phagocytosis, research is being directed towards the cellular envelope. By weakening the envelope, the bacteria may become more susceptible to attacks by the human immune system. Outer membrane integrity and structure is maintained in part by the Tol-PAL protein complex. This study focuses on TolB, a protein of this complex. PCR and sequencing were used to isolate and analyze the nucleotides that code for this protein from four strains of H. ducreyi. Phyre 2 was then used to determine the three dimensional structure of the protein. The amino acid sequences were identical for three of the four strains tested. The structure was also found to be comparable to the known TolB structure obtained from E. coli. This indicates that To lB is partially conserved across gram-negative bacteria. Further studies would help to clarify the importance of this conservation as related to protein structure and function.
Kinetic Studies of Glutathione S-Transferase
Tabitha Davis, 2012 (Advisor: Dr. Alice Deckert)
This study focuses on Glutathione S-Tranferase (GST) and the kinetics of the reaction it catalyzes: nucleophilic aromatic substitution. This enzyme is found in many different organisms including plants, animals, and humans and their basic function is to detoxify cells. The two binding sites of GST, the G site and the H site are specific for glutathione (GSH) and toxins (in this study, 2,4-dinitro-chlorobenzene (CDNB)), respectively. The catalyzed reaction was monitored at pH 6.5 using a stopped flow spectrophotometer to determine the rate constants for the reaction. The uncatalyzed reaction was monitored at pH 6.5 and 9 using an Ocean Optics UV-VIS spectrophotometer to compare to the catalyzed reaction. The uncatalyzed and catalyzed reactions showed similar kinetic profile shapes. By fitting the kinetic profiles with a bi-exponential (burst) fit (derived from the proposed mechanism) the uncatalyzed reaction showed very slow carbon-sulfur bond formation while the catalyzed reaction showed a much faster rate. The fitted data indicates that one of the rate constants is dependent on GSH concentration, while the other is not. By comparing the catalyzed and uncatalyzed reactions, it is apparent that GST is catalyzing both the ionization of GSH and the formation of the carbon-sulfur bond. The implications of this study in cancer research indicate that the ability to inhibit the ionization and stabilization power of the GST would inhibit its ability to metabolize chemotherapy agents.
The Enantiomeric Behavioral Effects of κ-opioid Agonist U-50488
Amon Manekul, 2012 (Advisor: Dr. Rodney Clark)
A recent study revealed the potency of U-50488, a κ-opioid agonist as an up-regulating agent of the immune system. The purpose of the present examine was to examine the differential effects the enantiomers of U-50488. Testing was done using 6 Sprague-Dawley rats on a Multiple, Fixed Ratio 10 Fixed Interval 90” schedule of water presentation, and sessions lasted for 25 minutes. Each enantiomer and the racemate were administered intraperitoneally in volumes of 1 mL/kg body weight and the dose varied from 0.3 mg/kg to 3.0 mg/kg. The results of the experiment show that (+) U-50488 is responsible for the longevity of response suppression whereas (-) U-50488 is responsible for the magnitude of response suppression.
A Thermodynamic Study of Multiple Single-Nucleotide Bulge Loops in RNA Duplexes
Eric Mastrogiacomo, 2012 (Advisor: Dr. Marty Serra)
RNA duplexes containing multiple single-nucleotide bulge loops were evaluated
via optical melting in 1M NaCl to determine the thermodynamic parameters (ΔHo, ΔSo, ΔG37o , and TM). Nine duplexes containing multiple bulge loops located in the same strand of the RNA duplex were analyzed. The distance of the bulge from the 5’ and 3’ end was varied while the inner duplex remained constant. Within the entire data set, the inner duplex varied in length as well as composition. The inner duplex was made up of 3 to 4 nucleotides and the identity of the nucleotides was varied as well. This thermodynamic data was added to previous research performed by Jones and compared to the revised Blose model (McCann et al., 2011). The revised Blose model does not accurately predict the effect of multiple single nucleotide bulges because the second bulge affects the duplex in a related but different manner than the first bulge. However, the revised Blose model does accurately predict that the bulge becomes less destabilizing as the stability of the less stable stem decreases. The trends found were not statistically different from each other, which is a result of the small data set. A larger data set of multiple nucleotide bulge loops should be studied in the future.
Structural and Thermodynamic Analysis of Group III Single Nucleotide Bulge Loops in a Hairpin in RNA and a Thermodynamic Analysis of Group IV Single Nucleotide Bulge Loops in Duplex in RNA
Michael McCann, 2012 (Advisor: Dr. Marty Serra)
Single nucleotide bulge loops occur when one nucleotide is unpaired in RNA duplexes. The bulge loops embedded in a hairpin studied in this investigation were of the group III variety, where ambiguity exists as to which nucleotide is bulged since the nearest neighbor can assume GU wobble base pairing instead of canonical Watson-Crick base pairing. The previous investigation of the thermodynamic parameters ∆H°, ∆S°, ∆G°37, and TM of thirty-five RNA duplexes containing group III single nucleotide bulge loops by optical melting in 1 M NaCl was completed and used to develop a model to predict the free energy of the destabilization of the duplex by the bulge. The thermodynamic data suggests that canonical Watson-Crick base pairing occurs instead of GU wobble base pairing, allowing a free energy model of group III bulge loops in duplex to be developed by treating them as group I bulge loops. The free energy model for predicting the stability of group I and group II bulge loops in a duplex is ΔGº37,bulge = -0.53 ΔGºstem + 0.7, where ΔGºstem is the least stable stem for group I bulge loops and is the second-least stable stem for group II bulge loops (McCann et. al, 2011). Since group III bulge loops can be considered group I bulge loops in a duplex if Watson-Crick base pairing takes place, single nucleotides of the group III variety can be predicted by the model. In this investigation, a structural analysis of RNA group III single nucleotide bulge loops embedded in a hairpin was utilized to complement the thermodynamic model of the stability of group III single nucleotide bulge loops in RNA duplexes and structurally solve the ambiguity. In-line structure probing of the hairpins with group III bulge loops suggest the position of the bulged nucleotide is the nucleotide further from the hairpin loop, which was similar to that of group II single nucleotide bulge loops embedded in a hairpin (McCann et al, 2011). The stability of hairpin sequences with group III single nucleotide bulge loops were compared to the stability model of hairpin sequences with group I and group II single nucleotide bulge loops to find that the group III bulge loops in a hairpin can predicted in the same manner that group I and II bulge loops in a hairpin are predicted. The destabilization of the hairpin structure with group I and group II bulge loops is related the stability of the stem opposite the hairpin from the bulge (Lim et al., 2012). Fourteen duplexes containing group IV single nucleotide bulge loops, which have a bulge environment that combines that of group II and III bulge loops, were optically melted in 1 M NaCl to obtain the thermodynamic parameters ∆H°, ∆S°, ∆G°37, and TM. The thermodynamic parameters were combined with a set of twenty-three duplexes with a group IV bulge loop to compare it to the model used to predict group I, II, and III single nucleotide bulge loops (McCann et al., in preparation). The free energy of any group of single nucleotide bulge loop in a duplex is ΔGº37,bulge = -0.65 ΔGºstem + 0.34 where the ΔGºstem is the ΔGº of the least stable stem for group I bulge loops, the ΔGº of the second least stable stem for group II and III bulge loops, and the ΔGº of the third least stable stem for group IV bulge loops.
Single Bulge Mutations Affecting the Rate of DNA:RNA Duplexing
Amanda McClelland, 2012 (Advisor: Dr. Alice Deckert)
DNA:RNA duplexes are significant in many biological processes. To understand these processes better, a simple system was created with sequences of DNA and RNA with varying parameters. Parameters include a bulge mutation, reactant concentration, and concentration of salt in solution. All three are important in the processes because the rate of duplex formation is affected based on the changes. In order to study this phenomenon, sequences of DNA and RNA were created with a single bulge mutation. The rates were observed using a Stopped-Flow instrument and the data was analyzed to determine if the mutation in the sequence affected the rate at which DNA and RNA form a duplex.
A Thermodynamic Investigation of the Stability of RNA-DNA Hybrid Duplexes Under Varying NaCl Conditions
Elizabeth McMichael, 2012 (Advisor: Dr. Marty Serra)
Thermodynamic parameters (ΔG, ΔH, ΔS, and TM) were determined for four RNA octomers, their homologous DNA strands, and complementary RNA-DNA hybrid duplexes through optical melting. The stability of duplex formation was investigated under varying NaCl concentrations ranging from 1.0M-0.01M. It has been previously shown that RNA-RNA duplexes are the most stable than corresponding DNA-DNA and RNA-DNA duplexes. The relative stability of DNA-DNA and DNA-RNA duplexes vary based on oligomer composition (Sugimoto et al., 1995). As expected, the RNA-RNA duplex had a greater stability than DNA-DNA duplex in 1.0M NaCl. In most cases, the stability of the hybrid duplexes fell between the RNA-RNA duplex and the DNA-DNA duplex. This study utilizes two sets of RNAs that differ in GC content, 62% to 75%. In all cases, the stability of the 75% GC content duplexes was greater than that of the 62% GC duplexes. The concentration of NaCl further destabilizes duplex formation in all of the oligomers, where greater thermodynamic stability is obtained at higher concentrations of NaCl . The overall purpose of this study is to obtain the thermal data of duplex formation for RNA-RNA, DNA-DNA, and RNA-DNA duplexes in order to compare to the kinetic data of duplex formation in the same duplexes under similar conditions.
Regulation of the HOX target gene CG13222 by Ultrabithorax
Olivia Mesoras, 2012 (Advisor: Dr. Brad Hersh)
HOX genes are found in all bilateral animals and are responsible for the regulation of anterior-posterior body patterning. HOX proteins regulate target genes by turning them on or off in specific tissues. The purpose of this study is to look at the regulation of the HOX target gene CG13222, which encodes a cuticle protein. CG13222 requires the HOX protein Ultrabithorax (UBX), which plays a role in haltere (hindwing) development, to bind its cis-regulatory element (CRE) for expression. Ultrabithorax requires two TAAT sequences in the CG13222 CRE for expression. However, this is likely not the only signal for UBX to bind, as TAAT is common in the genome. This study aimed to discover the other sequence information needed for UBX to bind to the CRE of CG13222. This was accomplished by mutating base pairs in the CRE near one required TAAT sequence. The mutations were generated using PCR sewing to mutate two base pairs at a time.
Analysis of Peptidoglycan Structure in Two Classes of Haemophilus ducreyi Using IR Spectroscopy and HPLC
Sarah Petrovich, 2012 (Advisor: Dr. Tricia Humphreys)
Haemophilus ducreyi is the Gram-negative bacterium that causes the sexually transmitted disease, chancroid. There are two populations of H. ducreyi, class I and class II, which vary in outer membrane proteins and therefore have different pathogenic properties and elicit different immune responses. Peptidoglycan is the main target of some antibiotics, such as vancomycin. Because vancomycin is a drug used primarily for the treatment of Gram-positive bacterial infections, it should not be sensitive to this antibiotic. Class II strains have been found to be sensitive to vancomycin, where class I strains are resistant, which suggests differences in the primary structure of peptidoglycan. Infrared spectroscopy was used to observe similarities and differences between the classes. IR spectroscopy only showed differences in the fingerprint region (1500-600 cm-1) of the spectra. High pressure liquid chromatography was also used, but the parameters were not able to be optimized and no peaks were obtained. It cannot be determined from the IR results if there are any significant differences in peptidoglycan structure between class I and class II H. ducreyi.
Computer Simulation Analysis for Electron Paramagnetic Resonance Spectroscopy Spectra of the Tetraheme Protein Cytochrome c554
Alicia Seggelink, 2012 (Advisor: Dr. Doros Petasis)
Cytochrome c554 is a tetraheme protein found in the nitrifying bacteria Nitrosomonas europaea and functions as an electron transport mechanism. The four Fe(III) ions, each coordinated within a Protoporphyrin IX ring, make up the active site of this metalloprotein. These iron ions are also paramagnetic species, meaning they have an unpaired electron that can interact with surrounding magnetic fields, whether they are from surrounding ligands and molecules or from an artificially applied magnetic field. This paramagnetic character makes this active site ideal for Electron Paramagnetic Resonance (EPR) spectroscopy. Initial studies of this protein have revealed the complexity of Cyt c554 in terms of its magnetic interactions and electrostatic behavior. In EPR, these characteristics lead to complicated spectra that cannot always be easily deciphered to extrapolate data and information about the electronic structure of the species under investigation. However, the use of computer simulations has been very helpful in terms of modeling a system.
This project focused mainly on refining a set of parameters that are used in the Spin-Hamiltonian, which is an operator that is used to define the possible energy states of a system as well as give information about the nature of the system. A program called SpinCount can evaluate the Spin-Hamiltonian according to the parameters that are input into the program to yield a simulation of those parameters, which will be an EPR spectrum. The goal of this study was to refine the parameter set until simulations visually agreed with the experimental spectrum of Cyt c554.
Three spectra were simulated that seem to correspond quite well with the experimental spectrum. According to the parameters used to create the simulations and the interactions that are observed, it is concluded that heme II and heme IV of Cyt c554 are ferromagnetically coupled and do not appear to have either strong nor weak coupling interactions. This investigation suggests that some intermediate level of coupling is occurring even though the parameters may hint towards weak coupling but the energy level diagrams and energy state transitions hint at a stronger coupling of the two heme irons. Further investigation of this system will certainly need to be conducted to fully characterize the electronic configuration and behavior of this heme protein, but the results obtained here could certainly be meaningful for that cause.
Kinetics of DNA Duplex Formation
Radek Stratil, 2012 (Advisor: Dr. Alice Deckert)
Kinetic profiles for renaturation of two DNA 10-mers (5’-GCATGTACGC/GCGTACATGC-3’ and 5’-GAAGGCTCTC/GAGAGCCTTC-3’) were recorded at a temperature range of 7ºC-35ºC and sodium chloride concentrations of 0.1M-1.0M using stopped-flow method. The profiles were fitted to a second-order integrated rate equation to derive the observed reaction rates. The change in entropy, ΔS⧧, and the change in enthalpy, ΔH⧧, of activation were derived from Eyring plots. The study was focused on determining the effect of alternating and stacked purine-pyrimidine sequences on the rate of renaturation. The ΔS⧧ for renaturation of the stacked strands decreased from approximately 25 kcal/molK at 0.01M NaCl to approximately 0 kcal/molK at 0.03M-1.0M NaCl indicating approximately equal levels of disorder in the single strands and the transition state. The ΔH⧧ followed the same trend with approximately 20 kcal/mol at 0.01M NaCl and 10 kcal/mol at 0.03M-1.0M NaCl. This trend indicates that at NaCl concentrations above 0.03M, the sodium ions shield the negative charges on the sugar-phosphate backbone decreasing the energy need for the association of the single strands to form the transition state. The ΔS⧧ for renaturation of the alternating strands was recorded to be approximately 40 kcal/molK for all sodium chloride concentrations indicating that the single strands are more highly organized than the transition state. This suggests the existence of a stable secondary structure formed by the single strands which was proposed to be a hairpin. The ΔH⧧ profile follows the same pattern as the ΔS⧧ and was recorded at approximately 21 kcal/mol for all the NaCl concentration indicating that there is substantial energy required to disrupt the secondary structure formed by the single strands in order to form the transition state which has lower order. Analysis of hyperchromicities indicated secondary structure in the alternating strand. The existence of this structure prevented comparison of the rates of renaturation between the alternating and stacked strands.
FbxA mediated ubiquitination of FbiA in Dictyostelium discoideum
Christine Wachnowsky, 2012 (Advisor: Dr. Margaret Nelson)
The highly conserved ubiquitin proteasome system (UPS) regulates the cell cycle, transcription factors and tumor suppressor proteins, like p53, by regulating protein levels. In order for ubiquitin mediated protein degradation to occur, the target must bind to an F-box protein, which is part of the Skp, Cullin, Fbox (SCF) complex of the E3 enzyme. Once bound, the target protein is polyubiquitinated and degraded by the proteasome. Since this process is highly conserved, it is often beneficial to study it in model organisms, such as Dictyostelium discoideum, in order to gain an understanding of the process that can be applied to more complex organisms. Two SCF complexes and their target proteins have been identified in D. discoideum¸ but a second potential target for the F-box protein FbxA has recently been identified as FbxA Interacting protein (FbiA). Based on this suggested interaction, I hypothesized that the FbiA protein fused to the reporter green fluorescent protein (GFP) should be ubiquitinated in wild type D. discoideum Ax2 cells as detected using immunopurification and Western analysis by probing with anti-GFP and anti-ubiquitin antibodies. I also hypothesized that by constructing an epitope-tagged version of ubiquitin, a more specific probe for ubiquitin can be used in the Western analysis to better detect ubiquitinated FbiA. Immunopurification and Western analysis demonstrated no detectable levels of bound protein when probed with anti-ubiquitin, so optimization of the protocol could enable detection. The plasmid was constructed using a vector containing a hemaglutinin tag and the D. discoideum gene for ubiquitin D; however further characterization of the plasmid and possibly the construction of a better plasmid will need to be conducted before it can be used to detect ubiquitinated FbiA. Continued analysis to look for ubiquitinated FbiA in D. discoideum can continue to provide insight into the role of the UPS in higher systems.
Identification of Glucose-inducible Indolic Metabolites Produced by Pseudomonas fluorescens
Amber Wetzel, 2012 (Advisor: Dr. Catharina Coenen)
Pseudomonas fluorescens is a root-associated bacterium that increases crop yield and protects plants against disease. In addition, some strains of P. fluorescens promote plant growth, most likely by colonizing roots and producing indole-3-acetic acid (IAA), the most common form of the plant growth hormone auxin. IAA production in P. fluorescens grown in Castric media supplemented with tryptophan, a precursor of IAA, can be quantified spectrophotometrically after reacting bacterial supernatants with the Salkowski’s reagent. Two biocontrol strains of P. fluorescens, Clinton and Eaton, produce IAA only when glucose is absent from the growth medium. In the presence of glucose, a different metabolite is produced, which also reacts with the Salkowski’s reagent, but has a different absorption maximum. To identify this metabolite, methods involving the analysis of products extracted with ethyl acetate from bacterial supernatant were being developed, including: further analysis with the Salkowski reagent, thin layer chromatography (TLC) and the van Urk-Salkowski’s reagent, and high performance liquid chromatography (HPLC). The conflicting results from these methods prevented the identification of unknown indole as any of the standard indoles: indole-3-acetonitrile (IAN), indole-3-pyruvid acid (IPyA), indole-3-acetaldehyde (IAAld), 1-methylindole (1-MI), and indole-3-carboxaldehyde (ICAld). Further analysis is important because the regulation of bacterial indole production by availability of plant-secreted sugars is an essential component of plant growth regulation by root-associated bacteria.
Interaction Between FbiA and FbxA in Dictyostelium discoideum
Michelle Williams, 2012 (Advisor: Dr. Margaret Nelson)
Dictyostelium discoideum is a valuable model system for the study of cellular processes, like cellular differentiation and cellular proportioning. Dictyostelium is thus used to observe the ubiquitin proteasome system (UPS), which functions through a pathway to target and mark proteins for degradation. FbxA is thought to be a F-box protein and functions in the UPS by specifically binding to target proteins. A yeast-two hybrid analysis suggests that FbxA targets FbiA for degradation (Christman, 1999). Previous research has attempted to further suggest that these two proteins interact by using co-immunoprecipitation to isolate FbxA and FbiA. However, these attempts have been unsuccessful. The goal of this study was to make modifications to the previous co-immunoprecipitation procedure, such as using a different negative control cell line, to successfully isolate FbxA and FbiA. A suggested interaction between FbxA and FbiA was seen in a co-immunoprecipitation of the experimental cell line, Ax2/Myc-FbxA;GFP-741. A co-immunopreciptation was not completed with the negative control cell line, Ax2/Myc-FbxA;GFP-αtubulin, due to complications developing these cells. Without a negative control, the suggestion that Myc-FbxA and GFP-741 interact is not definitive, for Myc-FbxA could be pulled out of solution independent of an interaction with GFP-741. In order to further determine if Myc-FbxA and GFP-741 interact in Dictyostelium discodium and further increase the efficiency of this co-immunoprecipitation, this experiment needs to be repeated for the negative control and modifications need to be made to the current experimental design.