Biology Commons^™

The Closing Mechanism Of Dna Polymerase I At Atomic Resolution, Bill R. Miller Iii, Loreena S. Beese, Carol A. Parish, Eugene Y. Wu

Biology Faculty Publications

Highlights

• DNA polymerase X-ray structure provides starting structure to investigate closing
• MD simulations provide atomistic details of DNA polymerase closing
• Propose sequence of events in prechemistry DNA polymerase active site assembly
• Identify a conserved histidine as a potential proton acceptor to initiate catalysis

Summary 
DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase …

Role Of Ime4 Protein In Pho Regulon Of S.Cerevisiae., Jenisha Ghimire

LSU New Orleans Theses and Dissertations

In the yeast Saccharomyces cerevisiae, the IME4 methyltransferase, interacts genetically with methyl binding protein, Pho92, to affect the expression of PHO regulon target genes. Cells mutant in IME4 or PHO92 show increases in the RNA abundance of PHO regulon target genes. The increase in the RNA abundance of the PHO regulon target genes is not additive in the cells double mutant in IME4 and PHO92. Hence, Ime4 and Pho92 interact in a single pathway in PHO regulon. Surprisingly, cells overexpressing IME4 and MUM2 shows increase in some PHO regulon target genes, indicating that IME4 affects the PHO regulon target …

A Novel Synthetic Yeast For Enzymatic Biodigester Pretreatment, Tianyu Tan, Mark S. Aronson, Arren Liu, Jill H. Osterhus, Melissa Robins, Suraj Mohan, Erich Leazer, Bowman Clark, Alexa Petrucciani, Katherine Lowery, James Welch, Casey Martin, Helena Lysandrou, Michael E. Scharf, Jenna Rickus

The Summer Undergraduate Research Fellowship (SURF) Symposium

Lignin, a complex organic polymer, is a major roadblock to the efficiency of biofuel conversion as it both physically blocks carbohydrate substrates and poisons biomass degrading enzymes, even if broken down to monomer units. A pretreatment process is often applied to separate the lignin from biomass prior to biofuel conversion. However, contemporary methods of pretreatment require large amounts of energy, which may be economically uncompelling or unfeasible. Taking inspiration from several genes that have been isolated from termites and fungi which translate to enzymes that degrade lignin, we want to establish a novel “enzymatic pretreatment” system where microbes secrete these …

Investigating The Interaction Of Aurka And Ube2c In Colorectal Cancer Cells, Apurva M. Hegde

Dissertations and Theses (Open Access)

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the US. Among the many genomic aberrations previously implicated in colorectal cancer, recurrent amplification of chromosome 20q is frequently associated with liver metastasis. Previous research in our lab identified a gene signature on chromosome 20q associated with colorectal cancer progression. In this study, one of the genes in the signature, the ubiquitin conjugating enzyme UBE2C, was identified through preliminary bioinformatics analysis as a candidate for further examination of its role in CRC progression. Co-expression analysis of UBE2C in tumor-normal datasets from the public database Oncomine revealed all the …

Treatment Strategies That Enhance The Efficacy And Selectivity Of Mitochondria-Targeted Anticancer Agents, Josephine S. Modica-Napolitano, Volkmar Weissig

Biology Faculty Publications

Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including …

Chronic Inflammation As A Result Of Hepatitis C Virus Infection: A Review Of The Literature, Samantha L. Lane

DePaul Discoveries

Approximately 170 million people are infected with Hepatitis C virus (HCV) worldwide^5,6. It is estimated that roughly 80% of those infected suffer from persistent infection with the virus; this persistence of infection is progressive, and over time can lead to fibrosis, cirrhosis, and hepatocellular carcinoma⁷. Chronic inflammation and apoptotic deregulation are both hallmarks of chronic HCV infection, and many molecular pathways are initiated in both the innate and adaptive immune responses during infection with this viral pathogen. The aim of this review was to survey some of the major molecular mechanisms responsible for the induction of …

Section Abstracts: Biology With Microbiology And Molecular Biology

Virginia Journal of Science

Abstracts of the Biology with Microbiology and Molecular Biology Section for the 93rd Annual Meeting of the Virginia Academy of Science, May 21-23, 2015, James Madison University, Richmond, Virginia

Characterization Of A Novel Clade Of Transporters In Phytophthora, Stephanie Padula, Paul F. Morris Dr, Howard Casey Cromwell Dr., Menaka Ariyaratne, Andrew Wagner

Honors Projects

The oomycete Phytophthora parasitica has a worldwide distribution and is an economically important pathogen of more than 100 species⁴. RNA-seq analysis showed that one gene, PPTG_16698 has the 5^th highest level of expression of all transport proteins in the zoospore stage, and is highly conserved throughout Phytophthora species. This project attempts to characterize the important biological role that PPTG_16698 plays in P. parasitica and other oomycetes. Three strategies have been implemented to accomplish this goal: growth analysis by heterologous expression in yeast, metabolite analysis in yeast, and construction of a GFP fusion protein to enable localization of …

Heterogeneous Dynamics In Dna Site Discrimination By The Structurally Homologous Dna-Binding Domains Of Ets-Family Transcription Factors, Gaofei He, Ana Tolic, James Bashkin, Gregory Poon

Chemistry & Biochemistry Faculty Works

The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only …

The Effect Of Red Maple Leaf Toxicosis On Reduced Glutathione Levels In Equine Erythrocytes In Vitro, Madeline A. Rohl

Undergraduate Honors Thesis Projects

Red maple leaf toxicosis is an equine blood disorder resulting from the consumption of wilted red maple (Acer rubrum L.) leaves by horses. Compounds within the leaves of red maple have oxidative effects on equine erythrocytes and can cause hemolysis of erythrocytes, the conversion of hemoglobin to methemoglobin, and the production of Heinz bodies. Reduced glutathione is important in the protection of equine erythrocytes from these oxidative events; however, in the presence of red maple toxin, glutathione is rapidly oxidized and is unavailable. The objective of this study is to determine whether the presence of vitamin C alters levels …

Analysis Of Chd Remodelers During Development: A Tale In Two Organisms, Brett Bishop

Open Access Dissertations

The correct development of different organisms requires the precise timing of genes important for development transitions. Organisms have recruited ATP-dependent chromatin remodelers to ensure the correct timing of gene expression during developmental transitions. Here I show how different CHD ATP-dependent chromatin remodelers regulate developmental transitions of different organisms. I show that PICKLE not only promotes H3K27me3 during development to repress developmental genes but also is targeted to these genes. The association of PICKLE to these genes suggests that both repression and H3K27me3 levels is a direct action of PICKLE on these loci. Using zebrafish as a model system, I show …

Conditional Regulation Of Puf1p, Puf4p, And Puf5p Activity Alters Yhb1 Mrna Stability For A Rapid Response To Toxic Nitric Oxide Stress In Yeast, Joseph Russo, Wendy Olivas

Biology Department Faculty Works

Puf proteins regulate mRNA degradation and translation through interactions with 3' untranslated regions (UTRs). Such regulation provides an efficient method to rapidly alter protein production during cellular stress. YHB1 encodes the only protein to detoxify nitric oxide in yeast. Here we show that YHB1 mRNA is destabilized by Puf1p, Puf4p, and Puf5p through two overlapping Puf recognition elements (PREs) in the YHB1 3' UTR. Overexpression of any of the three Pufs is sufficient to fully rescue wild-type decay in the absence of other Pufs, and overexpression of Puf4p or Puf5p can enhance the rate of wild-type decay. YHB1 mRNA decay …

A Functional Chlorophyll Biosynthesis Pathway Identified In The Kleptoplastic Sea Slug, Elysia Chlorotica, Julie A. Schwartz

USF Tampa Graduate Theses and Dissertations

The sacoglossan sea slug, Elysia chlorotica, feeds upon and sequesters plastids from the heterokont alga, Vaucheria litorea, and maintains the metabolically active organelles for up to nine months under starvation conditions while utilizing the photosynthate to survive and reproduce. The photosynthetic pigment, chlorophyll a (Chla), is found in all oxygenic photosynthetic organisms and is responsible for capturing photons of light and converting them into chemical energy. Chlorophyll and its associated proteins involved in the light capturing process are subject to photo oxidative damage and must be continually replaced for ongoing photosynthesis to continue; however, genes encoding these proteins are present …

Functional Characterization Of The Plant 15-Cis-Zeta-Carotene Isomerase Z-Iso, Jesus Alonso Beltran Zambrano

Dissertations, Theses, and Capstone Projects

Vitamin A deficiency is a widespread health issue in the tropics. To solve this issue, efforts are underway to increase provitamin A carotenoids such as β-carotene in staple crops which can be achieved by breeding, metabolic engineering or a combination of both approaches. However, rational strategies to improve carotenoid content in crops require sufficient knowledge of pathway regulation. Therefore, to better understand how plants synthesize provitamin A and to guide metabolic engineering strategies in crops such as maize, the functional characterization of the new ζ-carotene isomerase (Z-ISO) is of significant importance.

Z-ISO was recently discovered in maize and Arabidopsis (Chen …

Regulation Of Synaptogenesis By The Mirna Pathway And Fmr/P Bodies, Jacqueline Rochelle Furlong

Electronic Theses and Dissertations

Post-transcriptional regulation of mRNA is facilitated by different mechanisms, such as microRNA (miRNA) induced gene silencing or fragile X mental retardation protein (FMRP) mediated repression either independent of or acting through cytoplasmic RNA Processing bodies (P bodies). DPTP99A, Lar, and Wg have known functions during synaptogenesis and may be targets of miR-8. Here, we provide evidence that miR-8 regulates DPTP99A in vitro. Non-endogenous miR-8 expressed using an UAS driver regulates Lar. Endogenous miR-8 may regulate DPTP99A in vivo. Here we show that FMRP is capable of colocalizing with the P body components: DCP1, HPat, and Me31B, but not …

A Protective Role Of Autophagy In A Drosophila Model Of Friedreich's Ataxia (Frda), Luan Wang

Wayne State University Dissertations

Friedreich’s ataxia (FRDA) is an inherited autosomal recessive neurodegenerative disease. It affects 1 in every 50,000 people in central Europe and North America. FRDA is caused by deficiency of Frataxin, an essential mitochondrial iron chaperone protein, and the associated oxidative stress damages. Autophagy, a housekeeping process responsible for the bulk degradation and turnover of long half-life proteins and organelles, is featured by the formation of double-membrane vacuoles and lysosomal degradation. Previous researches indicate that Danon’s disease, the inherited neural disorder disease that shares similar symptoms with FRDA, is due to the malfunction of autophagy. Based on this, we raise the …

Rna Aptamers For Molecular Chaperones Hsp27 And Hsp90, Sathishkumar Kumar Munusamy

Legacy Theses & Dissertations (2009 - 2024)

Hsp90 and Hsp27 are members of the heat shock protein family of chaperones that perform multiple roles in cellular maintenance through protein folding and inhibition of apoptosis. They are abundantly expressed in cells and are over-expressed during conditions of stress. Hsp90 requires ATP for its chaperone function while Hsp27 self-associates into higher order oligomers enclosing its substrate. Their ability to interact with other proteins or with themselves lies at the heart of their mechanisms. The specific consequences of each of their interactions on global cellular health have not yet been fully discovered. The sheer diversity of proteins that interact with …

Genome-Scale Analyses Of Transcription And Transcriptional Regulation In Bacteria, Devon Marie Fitzgerald

Legacy Theses & Dissertations (2009 - 2024)

The textbook model of bacterial transcription regulation posits that promoters occur immediately upstream of genes and that transcription factors (TFs) modulate transcription through promoter-proximal binding. However, the recent application of unbiased genome-wide approaches, such as ChIP-seq and RNA-seq, has revealed a much more complex picture, including TF binding and transcription initiation occurring in unexpected locations. This dissertation describes the use of deep sequencing-based approaches to evaluate the genome-wide binding of transcription-related proteins and identify locations of transcription initiation. I have assessed the genome-wide binding of three Escherichia coli TFs and an alternative σ factor. Additionally, I have analyzed genome-wide patterns …

Cross-Talk Between The Tumor Suppressors Par-4 And P53, Tripti Shrestha Bhattarai

Theses and Dissertations--Toxicology and Cancer Biology

This work describes the fascinating interplay between two tumor suppressors Prostate apoptosis response-4 (Par-4) and p53. The guardian of the genome, p53, is frequently mutated in human cancers, and may contribute to therapeutic resistance. However, p53 is intact and functional in normal tissues, and we observed that specific activation of p53 in normal fibroblasts could induce apoptosis selectively in p53-deficient cancer cells. This paracrine apoptotic effect was executed by Par-4 secreted in response to p53 activation. Accordingly, activation of p53 in wild-type mice, but not in p53^-/- or Par-4^-/- mice, caused systemic elevation of Par-4 that induced apoptosis …

Functional Analysis Of The Ovarian Cancer Susceptibility Locus At 9p22.2 Reveals A Transcription Regulatory Network Mediated By Bnc2 In Ovarian Cells, Melissa Buckley

USF Tampa Graduate Theses and Dissertations

GWAS have identified several chromosomal loci associated with ovarian cancer risk. However, the mechanism underlying these associations remains elusive. We identify candidate functional Single Nucleotide Polymorphisms (SNPs) at the 9p22.2 ovarian cancer susceptibility locus, several of which map to transcriptional regulatory elements active in ovarian cells identified by FAIRE-seq (Formaldehyde assisted isolation of regulatory elements followed by sequencing) and ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) in relevant cell types. Reporter and electrophoretic mobility shift assays (EMSA) determined the extent to which candidate SNPs had allele specific effects. Chromosome conformation capture (3C) reveals a physical association between Basonuclin 2 (BNC2) and …

High-Throughput Screening Of Age-Related Changes In Caenorhabditis Elegans, Neil Copes

USF Tampa Graduate Theses and Dissertations

This project was developed to identify novel methods for high-throughput culturing and screening of C. elegans to investigate age-related metabolic changes and to survey the proteomic and metabolomic factors associated with age-related changes. To accomplish these goals we developed a novel way to grow C. elegans in liquid culture in 96-well microplates for several weeks without suffering significant fluid loss due to evaporation and without needing to shake or unseal the plates for aeration. We also developed methods for assaying the total volume of live C. elegans in microplate cultures using a fluorescence microplate reader and for performing RNAi experiments …

The Effects Of Supplemented Metabolites On Lifespan And Stress Response Pathways In Caenorhabditis Elegans, Clare B. Edwards

USF Tampa Graduate Theses and Dissertations

Understanding how metabolites contribute to anaplerosis, antioxidant effects, and hormetic pathways during aging is fundamental to creating supplements and dietary habits that may decrease age-associated disease and decline, thus improving the quality of life in old age. In order to uncover metabolic pathways that delay aging, the effects of large sets of metabolites associated with mitochondrial function on lifespan were investigated.

Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in C. elegans. Addition of fumarate and succinate also extended lifespan and all three metabolites activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced …

Drosophila Cyclin J And The Somatic Pirna Pathway Cooperate To Regulate Germline Stem Cells, Paul Michael Albosta

Wayne State University Dissertations

Cyclin J (CycJ) is a highly conserved cyclin that is uniquely expressed specifically in ovaries in Drosophila. Deletion of the genomic region containing CycJ and adjacent genes resulted in a genetic interaction with neighboring piRNA pathway gene, armitage (armi). Here I assessed oogenesis in CycJ null in the presence or absence of mutations in armi or other piRNA pathway genes. Although CycJ null flies had decreased egg laying and hatching rates, ovaries appeared normal indicating that CycJ is dispensable for oogenesis under normal conditions. Further double mutant analysis of CycJ and neighbor armi, as well as two other piRNA pathway …

Analyzing The Interactions Of Kdm5/Lid And Sin3 In Drosophila Melanogaster, Ambikai Gajan

Wayne State University Dissertations

SIN3, the scaffold protein of a histone modifying complex is conserved from yeast to mammals. Drosophila SIN3 associates with both a histone deactylase RPD3 and a histone demethylase dKDM5/LID. Immunopurification of dKDM5/LID verifies a previously observed interaction with SIN3 and RPD3. Furthermore, deficiency of dKDM5/LID phenocopies deficiency of SIN3 in many cellular and developmental processes. Knockdown of both Sin3A and lid hinder cell proliferation in Drosophila cultured cells and developing flies. Knockdown of these genes also results in a curved wing phenotype implicating a role in wing development. Analysis of underlying gene expression changes upon decreased expression of SIN3, dKDM5/LID …

Mysteries Of The Trypanosomatid Maxicircles: Characterization Of The Maxicircle Genomes And The Evolution Of Rna Editing In The Order Kinetoplastida, Preethi Ranganathan Iyengar

Theses and Dissertations

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates.

This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 …

Modeling Atp-Binding Cassette G2 (Abcg2) Substrate Specificity, Raghav D. Acharya, Aurijit Sarkar, Glen E. Kellogg

UROP Posters

How well can we predict efflux by ATP-binding cassette G2?

It is estimated that there will be about 1.6 million new cases of cancer and half a million cancer deaths in the US during 2015.ATP-binding cassette (ABC) efflux transporters such as ABCG2 are overexpressed in chemotherapy-resistant cancer cells. Anticancer drugs are prone to efflux by these transporters. Being able to identify drugs that are effluxed is of great interest in drug discovery.The current arsenal of methods used to detect efflux are not easily adaptable to high throughput formats and are limited in scope, making experimental analysis an expensive prospect. Hence, …

A Conservative Isoleucine To Leucine Mutation Causes Major Rearrangements And Cold Sensitivity In Klentaq1 Dna Polymerase, Eugene Y. Wu, Amanda R. Walsh, Emma C. Materne, Emily P. Hiltner, Bryan Zielinski, Bill R. Miller Iii, Lily Mawby, Carol A. Parish, Wayne M. Barnes, Milko B. Kermekchiev

Biology Faculty Publications

Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary …

Purification And Characterization Of Bcsc; An Integral Component Of Bacterial Cellulose Export, Emily D. Wilson Ms

Theses and Dissertations (Comprehensive)

Biofilms are a growing concern in the medical field due to their increased resistance to antibiotics. When found in a biofilm, bacteria can have antibiotic resistance 10-1000 times that of their planktonic counterparts. Therefore, it is important to study the formation of biofilms. Cellulose biofilms are formed by Enterobacteriaceae, such as many Escherichia coli and Salmonella spp. strains. Biofilms provide these species with benefits including antimicrobial protection, development of bacterial communities, promotion of DNA exchange, uptake of nutrients, and, in the case of cellulose biofilms, immune system evasion. Cellulose biofilms are controlled by the Bacterial cellulose synthesis (Bcs) complex located …

Characterization Of A Putative Phospholipase D ´ Like Gene As A Lipid Signaling Modulator And Its Role In Salicylic Acid Mediated Defense Pathway In Nicotiana Tabacum, Phillip T. Dean

Electronic Theses and Dissertations

Plants are in a perpetual evolutionary arms race with a wide range of pathogens. Their sessile nature has led plants to evolve defense mechanisms that can quickly recognize a unique stressor and deploy a resistance tailored for a specific attack. The salicylic acid (SA) mediated defense pathway has been shown to be one of the major defense tactics plants can initiate to defend themselves against microbial pathogens. Following a pathogen attack high levels of methyl salicylate (MeSA) are produced that can be converted to SA by the enzyme salicylic acid binding protein 2 (SABP2). A yeast two-hybrid screening was performed …

Ribosomes Left In The Dust: Diverse Strategies For Peptide-Mediated Translation Stalling, Benjamin H. Hudson, Hani S. Zaher

Biology Faculty Research

In two recent papers, Arenz et al. (2014a) and Bischoff et al. (2014) provide structural insights into drug-induced, peptide-mediated stalling of the ribosome.