Students in biology

SUMMER RESEARCH PROGRAM

2012 Student Research Abstracts 

Biology Summer Research group

 

Alyson Smith, Marquette University

Mentor: Dr. Kathleen Karrer

Construction of a Kinase Knockout Plasmid for Tetrahymena thermophila

 

Hannah Zimmerman, Wesleyan University

Mentor: Dr. Gail Waring

The Effect of a Dominant Negative dec-1 Trangene on Wild Type dec-1 Eggshell Proteins

 

Julien Aoyama, Amherst College

Mentor: Dr. Allison Abbott

Genetic Analysis of the Defecation Motor Program Downstream of mir-786 in C. elegans

 

Thomas Yates, Covenant College

Mentor: Dr. Pinfen Yang

Differential Deficiency of the Radial Spoke Along the Length of the Flagellar Axoneme of Chlamydomonas Mutants

 

Michelle Evans, Brown University

Mentor: Dr. James Buchanan

The Locomotor Network in the Lamprey Hindbrain: Spinal Input, Oscillatory Mechanisms, and Neuromodulation

 

Maxwell DeNies, SUNY Fredonia

Mentor: Dr. Krassimira Hristova

The Human Impact on Antibiotic Resistance

 

Jonathan Hanna, Marquette University

Mentor: Dr. James Maki

How Does Surface Charge and Cetylpyridinium Chloride (CPC) Affect the Microbial Diversity on Dental Materials?

 

Scott Hetzel, Ripon College

Mentor: Dr. Rosemary Stuart

Functional Importance of Disulfide Bond Formation in Cox12 and the Possible Role of Rcf1/ Rcf2 Proteins

 

Joseph Burclaff, Marquette University

Mentor: Dr. James Anderson

Analyzing the Effects of K904N and V1019A Point Mutations on the Activity of Mtr4

 

Melissa Budelier, Marquette University

Mentor: Dr. Martin St. Maurice

Investigating the Intramolecular Interaction of the Biotin Carboxyl Carrier Domain with the Carboxyl Transferase Domain in Pyruvate Carboxylase

 

  

 

 

Alyson Smith, Marquette University

Mentor: Dr. Kathleen Karrer

Construction of a Kinase Knockout Plasmid for Tetrahymena thermophila

Tetrahymena thermophila is a protozoan with two nuclei, a Micronucleus (Mic) and a Macronucleus (Mac). During mating, the parental Mac is degraded and a new Mac develops. The developing Mac (which is called an Anlagen) goes through a process called endocycling where it replicates its DNA several times. The Tetrahymena gene ASI2 (Anlagen Stage Induced) is up regulated during mating as is required for endocycling in the developing Mac. Germ line (Mic) knockouts of ASI2 do not make viable progeny. The working hypothesis, based on in silico analysis and site directed mutagenesis, is that Asi2p is a signal transducer that needs to be phosphorylated for it to function. TTHERM_00717590 is a putative kinase that is expressed at the same time in mating as ASI2. To determine whether TTHERM_00717590 is involved in endocycling, a construct was created to knock this gene out in Tetrahymena via homologous recombination. Plasmid DNA of the vector pNeo4 was prepared. The plasmid pNeo4 contains the selectable neo gene which was optimized for Tetrahymena codon usage. Mochizuki, K (Gene 425:79(2008)).PCR primers were designed and genomic PCR was optimized to obtain 5' and 3' fragments of the gene of interest. The 5' fragment was ligated into the 5' MCS of pNeo4 and the resulting plasmid was transformed into SURE cells. Plasmid DNA of the pAck5 (ASI2 co-expressed kinase) construct was isolated and characterized by restriction enzyme digestion and DNA sequencing. Experiments are in progress to obtain transformants with the 3' PCR fragment inserted into pAck5. The long-term goal of this project is to use the construct pAck35 to knock out TTHERM_00717590 in Tetrahymena.

 

Hannah Zimmerman, Wesleyan University

Mentor: Dr. Gail Waring

The Effect of a Dominant Negative dec-1 Trangene on Wild Type dec-1 Eggshell Proteins

The dec-1 protein is essential for proper eggshell assembly in Drosophila. The dec-1 gene is alternatively spliced to form three different sized proproteins: 177kDa, 125 kDa, and 106 kDa. During eggshell assembly, the major product, fc106 is cleaved during specific stages. The Δs20 transgene, a dec-1 transgene in which the s20 region is deleted, has a dominant-negative effect on female sterility. When the Δs20 transgene is introduced into wild type females, 100% of the females are sterile. In order to investigate how the Δs20 mutation exerts its dominant negative effect, a His-tagged dec-1 transgene was created to track wild type dec-1 derivatives. A 6x-His tag was inserted into the unique C-terminus of the fc 106 proprotein. RT-PCR and Western blot analyses suggest the transgene is expressed at both the RNA and protein levels. However, small dec-1 derivatives, rather than the major dec-1 cleavage products were found, suggesting that fc106 undergoes early C-terminal processing. Thus, the dec-1 -His transgene has limited usefulness for tracking dec-1 derivatives. Previous studies using an antibody directed against the fc106 C-terminal region, cfc 106, showed dec-1 products in the chorion and vitelline membrane layers in late stage 14 egg chambers. Immunofluorescence confocal microscopy was used to visualize dec-1 products in flies with the dominant negative Δs20 transgene. Ovarian serial sections were stained with either anti-s18 (a chorion layer marker) or the cfc106 antibody. The images suggest that dec-1 products localize exclusively to the main shell region until late stage 14, when dec-1 products are also found in the dorsal appendages, specializations of the chorion layer. Thus it appears that late localization of dec-1 products in the chorion is not affected by the dominant negative Δs20 transgene.

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Julien Aoyama, Amherst College

Mentor: Dr. Allison Abbott

Genetic Analysis of the Defecation Motor Program Downstream of mir-786 in C. elegans

Defecation is a calcium-dependent rhythmic behavior in C. elegans and repeats about every 50 seconds in wild-type worms. The defecation cycle is triggered by an intercellular calcium wave that initiates in the posterior intestine. mir-786 encodes a microRNA that is required for rhythmic defecation behavior, as mir-786 mutant worms have long, arrhythmic defecation cycles and defects in calcium wave initiation in the posterior intestine. miR-786 regulates the translation of elo-2, a fatty acid elongase whose levels are conversely correlated with those of the fatty acid palmatate. We propose that miR-786 functions to repress elo-2 to promote the palmitolation of the regulatory membrane proteins, such as the TRPM channels, GON-2 and GTL-2, specifically in the posterior intestine to help initiate the calcium wave. We also propose phospholipase C β (egl-8) is involved in propagation of the calcium signal once it is initiated in the posterior intestine. To test this model, I performed genetic epistasis experiments. I found that the mir-240/786 mutant phenotype requires gtl-1 activity, consistent with mir-786 functioning upstream of gtl-1 in the regulation of defecation behavior. I also found that loss of egl-8 enhanced the defecation defects of mir-240/786 mutants. Future work will examine the remaining 11 candidate palmitoyl transferase genes that haven't been tested with RNAi experiments. Palmitolyation of targets in the posterior intestinal cells may be essential for conferring supremacy to the posterior intestinal cells and thus knockdown on the essential palmitoyl transferases may phenocopy the mir-786 phenotype in wild-type worms. Characterization of the calcium signaling differences between mutant and wild-type worms will also be examined by use of a calcium biosensor that fluoresces when bound to calcium ions in live worms.

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Thomas Yates, Covenant College

Mentor: Dr. Pinfen Yang

Differential Deficiency of the Radial Spoke Along the Length of the Flagellar Axoneme of Chlamydomonas Mutants

One major question regarding eukaryotic cilia and flagella is how the intricate macromolecular supercomplex, the axoneme, is assembled in these slender organelles. In a typical 9+2 axoneme, a number of molecular complexes associate with microtubules at particular locations repetitively throughout the length of flagella to stabilize microtubules and to drive rhythmic beating. The assembly process is elaborate and tightly regulated, as individual polypeptides in each axonemal complex are exclusively synthesized in the cell body, most of which are then prepackaged into precursor complexes and delivered by the intraflagellar transport train into flagella to be added to the tip of the growing axoneme after cell division. To elucidate this process, we investigated two mutant strains of the biflagellate Chlamydomonas, pf5 and pf27. Most of these mutant cells have paralyzed flagella with reduced amounts of axonemal complex, the radial spoke and spoke proteins, which are hypo-phosphorylated. By expressing a green florescent protein (GFP)-tagged spoke protein, we discovered that the fluorescing spoke primarily enriched at the base of flagella, with a strong intensity in pf5, whereas RSs distribute along the entire length of wild type flagella. Interestingly, expression of the fusion protein, without correcting the genetic deficiency, increased the percentage of swimmers, albeit with aberrant trajectory. These data suggest that the assembly process changes as flagella elongate; the mutations in pf5 and pf27 cause RS assembly efficiency, especially toward the end of the assembly process. Additional GFP-RSPs increase the amounts of assembled RSs above the threshold level enabling more cells to swim with partially restored motility. We hypothesis that hypo-phosphorylation in pf5 and pf27 prevents RSP3-GFP from incorporating at a rate complementary to microtubules as flagella elongate; we believe that this proximal isolation of the RS in transformed pf5 and pf27 is responsible for the aberrant swimming patterns observed in these strains.

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Michelle Evans, Brown University

Mentor: Dr. James Buchanan

The Locomotor Network in the Lamprey Hindbrain: Spinal Input, Oscillatory Mechanisms, and Neuromodulation

It is well-established that the neural network responsible for generating the rhythmic motor pattern of locomotion exists in the spinal cord of all vertebrates. This network is called the locomotor central pattern generator (CPG). However, recent evidence indicates that the CPG also exists within the hindbrain of lower vertebrates. Our main questions are whether or not this hindbrain locomotor network can be active without spinal cord input and whether this hindbrain network shares similar mechanisms with the spinal CPG. In order to answer these questions, we used an isolated brainstem-spinal cord preparation of the adult lamprey, a lower vertebrate. We activated the locomotor CPG by bathing the preparation in D-glutamate, the main excitatory neurotransmitter. Locomotor activity was recorded with extracellular suction electrodes placed on motor nerves such as the spino-occipital nerves of the hindbrain and the ventral roots of the spinal cord. The membrane potentials of individual motor neurons were recorded with sharp intracellular microelectrodes. To allow independent control of the brainstem and spinal cord bathing solutions, a Vaseline diffusion barrier was constructed at the hindbrain-spinal cord junction. When we added high divalent cation solution to the spinal cord, which suppressed its activity, we found that although the brainstem was still rhythmically active, the amplitude of spino-occipital motor neuron oscillations was reduced by 60%. Thus, spinal input to the brainstem enhances rhythmic activity in the hindbrain but is not necessary for the hindbrain rhythm. We then asked whether the hindbrain CPG exhibits similar neural mechanisms as the spinal CPG by applying drugs to the brainstem bath when spinal cord activity was suppressed. Similar to the spinal CPG, strychnine, a blocker of inhibitory glycine receptors, converted the normal alternating bursting across the midline to synchronous bursting. Also similar to the spinal CPG, ZD7288, a blocker of the hyperpolarization-activated cation current (Ih), slowed the locomotor burst rate. Finally, serotonin, a neuromodulatory transmitter, slowed the locomotor burst rate in the hindbrain, similar to its effects in the spinal cord. In conclusion, the lamprey hindbrain has a locomotor CPG that can function without spinal input and shares several mechanisms with the spinal locomotor CPG.

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Maxwell DeNies, SUNY Fredonia

Mentor: Dr. Krassimira Hristova

The Human Impact on Antibiotic Resistance


An increasing prevalence of antibiotic resistant microbes threatens our current ability to treat bacterial infections. Although antibiotics are characterized by a fundamental flaw – use leads to decreased efficacy – human activities perpetuate this progression. The primary goal of this study was to determine the impact of Waste Water Treatment Plants (WWTPs) on environmental antibiotic resistance. E. coli were collected from three unique environments: a clinical setting (Medical College of Wisconsin), human impacted areas (influent and effluent WWTPs and polluted rivers), and minimally human impacted locations (Lake Michigan). Isolates were tested for resistance to 12 antibiotics from 9 unique antibiotic classifications. Isolate screening was performed in 96 well plates with clinically therapeutic antibiotic dosages. Antibiotic concentration was diluted in subsequent wells. Isolate resistance was determined via turbidity at specific antibiotic concentrations. Turbidity at the highest antibiotic concentration defined resistance while little or no growth at lower concentrations defined intermediate resistance or sensitivity. Additionally, PCR was used to identify common resistance genes and increase the robustness of the study. Principle Component Analysis and Hierarchical Clustering were used to discover patterns of resistance and attain greater understanding of the impact of WWTPs on antibiotic resistance. Isolates showing multi-resistance (resistance to greater than 3 antibiotic classifications) was significantly greater (p < 0.05) in effluent than influent water. Independent of location non-clinical isolates (human impacted and minimally human impacted) had highest resistance to ampicillin, erythromycin, and sulfamethoxazole. Comparatively, clinical isolates had higher resistance to aminoglycosides comparatively. Additionally, clinical isolates had no resistance to fosfomycin while resistance was common among non-clinical complements. Overall influent isolates had highly variable resistance profiles, while effluent bacteria were ubiquitously highly resistant (PCA). These results suggests that, with respect to E. coli, WWTPs create a selective environment for resistant bacteria and/or increase the transferability of resistance elements between bacteria. An overall better understanding of antibiotic resistance, the mechanisms of resistance, transmission of resistance genes, and factors leading to the increased prevalence of antibiotic resistant elements in non-clinical settings is essential for improvement or mere retention of our current level of public health and effective disease treatment possibilities.

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Jonathan Hanna, Marquette University

Mentor: Dr. James Maki

How Does Surface Charge and Cetylpyridinium Chloride (CPC) Affect the Microbial Diversity on Dental Materials?

Dental materials serve to increase one’s oral health.  However, these foreign materials serve as additional attachment sites for oral microbes.  This project used microbes in natural saliva to 1) explore the diversity of biofilm microbes attached to charged and uncharged denture materials, and 2) how adding cetylpyridinium chloride (CPC) to denture materials affected the biofilm diversity. After exposure to saliva that contained natural microbes, DNA was extracted from biofilms established on charged and uncharged poly-methyl(methacrylate) (PMMA) disks and partial 16S ribosomal RNA genes from the community were amplified using the polymerase chain reaction (PCR).  Biofilm diversity on the various substrata was examined by fingerprinting the bacterial community using denaturing gradient gel electrophoresis (DGGE).  The DGGE data indicated that there was no difference in the diversity of attached bacteria on charged and uncharged substrata.  However, treatment of substrata with CPC indicated that there was a difference in the microbial diversity between CPC and non-CPC treated disks.  A preliminary experiment showed that pretreating artificial substrata with CPC inhibited attachment by the oral bacterium, Enterococcus faecalis for 2 hours of exposure (P<0.001).  The data after 24 hours indicates that CPC does have an effect on the microbial diversity.  Sequencing of the different bands would provide identification of the microorganisms involved.  The effect of CPC on the diversity at shorter time periods is unknown and needs to be addressed in future experiments.

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Scott Hetzel, Ripon College

Mentor: Dr. Rosemary Stuart

Functional Importance of Disulfide Bond Formation in Cox12 and the Possible Role of Rcf1/ Rcf2 Proteins

The cytochrome c oxidase complex (COX), a multisubunit complex, is a key enzyme of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. Saccharomyces cerevisiae subunit Cox12, encoded by nuclear DNA, is a COX subunit involved in the binding site for cytochrome c to the COX complex. A deficiency in Cox12 assembly affects the enzyme efficiency of the COX complex, thus significantly decreasing the level of OXPHOS activity. Rcf1, respiratory supercomplex factor 1, is a novel protein that may act as a bridge between the cytochrome bc1 and the COX enzyme in their supercomplex organization. Rcf1 shares overlapping function with Rcf2, which is required for optimal COX enzyme activity and for the assembly of the Cox12 subunit with the COX complex. The crystal structure of the bovine homolog of the Cox12 protein, the Cox6b protein, indicates the presence of two intramolecular disulfide bridges. In the yeast Cox12 protein, these bridges occur between the highly conserved Cys27 and Cys59 and the Cys37 and Cys48 residue pairs. The functional relevance of these intramolecular disulfide bridges is unknown however, and was investigated here. Using thiol-alkylating reagent 4-acetamido-4’-maleimidylstilbene-2, 2’-disulfonic acid (AMS), we could demonstrate that Cox12’s disulfide bonds are formed in the absence of Rcf1 and Rcf2 and thus may not account for the lack of Cox12 assembly in the absence of these proteins. To directly test the importance of the disulfide bonds for Cox12 function, the COX12 gene was cloned into yeast integrating plasmid (Yip-351) under the control of the Gal10 promoter and site-directed mutagenesis was performed to mutate the cysteine (C) residues to serine (S), thereby generating cox12C27S and cox12C48Ssingle mutants, as well as a cox12C27S, C48S double mutant. Following sequencing verification, the plasmids harboring the mutant cox12 genes were transformed into Δcox12 mutant yeast cells and complementation analysis was performed. Results to demonstrate that formation of both disulfide bonds is not essential for Cox12 function will be presented here.

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Joseph Burclaff, Marquette University

Mentor: Dr. James Anderson

Analyzing the Effects of K904N and V1019A Point Mutations on the Activity of Mtr4

The Mtr4 protein functions as part of the TRAMP (Trf4/Air2/Mtr4p Polyadenylation) complex, which is active in RNA surveillance and degradation. Mtr4 is known to act as an RNA helicase, hydrolyzing ATP to fuel RNA unwinding, yet much about it remains unknown, such as the purpose of the novel arch domain and the c-terminal end of the protein. By creating mutations in the c-terminal end and analyzing their effects on yeast growth and the proteins’ enzymatic abilities, these regions of the Mtr4 protein can be further characterized and understood. Phenotypic expression tests indicated that the K904N and V1019A point mutations significantly affect the functioning of Mtr4, so focus was given to them for the project. In an attempt to analyze the enzymatic effects of the mutations, both were purified by column chromatography over a charged nickel resin and Fast Protein Liquid Chromatography (FPLC). Apparent problems in the nickel binding and FPLC resulted in impure samples with inconclusive assay results, so future work will focus on improving the collection and purification of the mutant proteins. Following the results of the phenotypic expression, a northern blot was performed on both mutations and control strains to detect initiator tRNA, or tRNAiMet, to test the hypothesis that Mtr4 mutant strains will degrade tRNAiMet less efficiently. The northern blot suggested that the mutations actually resulted in lower tRNAiMet levels, yet more work needs to be done due to variance and error in the procedure. In conclusion, while the exact effects of the K904N and V1019A point mutations remain to be determined, these results indicate that the c-terminal end of Mtr4 is integral to its proper functioning within the TRAMP complex.

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Melissa Budelier, Marquette University

Mentor: Dr. Martin St. Maurice

Investigating the Intramolecular Interaction of the Biotin Carboxyl Carrier Domain with the Carboxyl Transferase Domain in Pyruvate Carboxylase

 

Pyruvate Carboxylase (PC) is a tetrameric protein that catalyzes the formation of oxaloacetate (OAA) via a two-step mechanism.  In the first catalytic step, biotin is carboxylated in the biotin carboxylase (BC) domain.  The biotin carboxyl carrier protein (BCCP) subsequently transfers the carboxybiotin from the BC domain to a neighboring carboxyl transferase (CT) domain on a physically separate polypeptide.  The CT domain then catalyzes the transfer of the carboxyl group from carboxybiotin to pyruvate, forming OAA [St Maurice et al, 2007]. When recombinant PC is expressed as a dimer, a neighboring CT domain is not present to accept the BCCP domain.  Previous research has shown that this recombinant PC still exhibits surprisingly high activity despite its inability to access a neighboring CT domain [Lietzman et al, 2011].  This suggests the possibility that PC is capable of an intermolecular BCCP-CT domain interaction.  In order to address this, we have assed activity levels of recombinant hybrid dimers with various combinations of inactive BCCP and CT domains.  In doing so, we have gained insight into the intermolecular function of the CT domain.

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