Life Sciences Commons^™

Fluorescence-Reported Allelic Exchange Mutagenesis Reveals A Role For Chlamydia Trachomatis Tmea In Invasion That Is Independent Of Host Ahnak, M. J. Mckuen, Konrad E. Mueller, Y. S. Bae, Kenneth A. Fields

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Development of approaches to genetically manipulate Chlamydia is fostering important advances in understanding pathogenesis. Fluorescence-reported allelic exchange mutagenesis (FRAEM) now enables the complete deletion of specific genes in C. trachomatis L2. We have leveraged this technology to delete the coding sequences for a known type III effector. The evidence provided here indicates that CT694/CTL0063 is a virulence protein involved in chlamydial invasion. Based on our findings, we designate the gene product corresponding to ct694-ctl0063 translocated membrane-associated effector A (TmeA). Deletion of tmeA did not impact development of intracellular chlamydiae. However, the absence of TmeA manifested as a decrease in infectivity …

Mutagenesis Of Human Alpha-Galactosidase A For The Treatment Of Fabry Disease, Erin Stokes

Dissertations, Theses, and Capstone Projects

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40-60 years of age. There is currently only one FDA approved treatment (Fabrazyme) classified as an enzyme replacement therapy. However, approximately 88% of patients experience a severe immune response that, rarely, can be fatal and is a huge cost burden at average $250,000 a year per patient. The structure of α-galactosidase A has been previously determined to be a …

Identification Of Deletions In The Hdc Gene Of Drosophila Melanogaster Generated Through Transposon-Excision Mutagenesis, Gregory A. Wesseling

Masters Theses

Histamine is a biogenic amine that functions as a neurotransmitter in a number of vertebrate and invertebrate systems and is synthesized from its precursor histidine by the enzyme histidine decarboxylase (HDC). In Drosophila, histamine has been shown to have function in photoreceptors, mechanoreceptor cells, as well as centrally located neurons. Mutations of the Hdc gene, such as Hdc^JK910, exhibit defects in histamine synthesis and display altered behaviors such as blindness, inability to groom, impaired thermal tolerance, and altered sleep rhythms. However, all Hdc mutants obtained thus far demonstrate some transcriptional activity.

In order to remove Hdc expression …

Determination Of Amino Acids Involved In Specificity And Activity Of Chladub2, Trent S. Arbough, John M. Hausman, Chittaranjan Das

The Summer Undergraduate Research Fellowship (SURF) Symposium

Chlamydia trachomatis is a pathogen which infects humans as a sexually transmitted disease or through ocular infection, causing ocular trachoma. Ocular trachoma is the leading cause of non-congenital blindness in developing countries. The bacteria employs the deubiquitinating enzyme ChlaDUB2 to remove ubiquitin from its inclusion membrane in order to avoid lysosomal degradation. Key amino acids involved in ubiquitin recognition and cleavage were mutated in order to probe substrate specificity and catalytic activity of ChlaDUB2. Mutants were used in fluorometry assays in order to determine how the mutations affect the ability of ChlaDUB2 to release the amino methyl coumarin (AMC) group …

Paracrine Regulation Of Melanocyte Genomic Stability: A Focus On Nucleotide Excision Repair, Stuart Gordon Jarrett, Katharine Marie Carter, John August D'Orazio

Markey Cancer Center Faculty Publications

UV radiation is a major environmental risk factor for the development of melanoma by causing DNA damage and mutations. Resistance to UV damage is largely determined by the capacity of melanocytes to respond to UV injury by repairing mutagenic photolesions. The nucleotide excision repair (NER) pathway is the major mechanism by which cells correct UV photodamage. This multistep process involves the basic steps of damage recognition, isolation, localized strand unwinding, assembly of a repair complex, excision of the damage‐containing strand 3′ and 5′ to the photolesion, synthesis of a sequence‐appropriate replacement strand, and finally ligation to restore continuity of genomic …

Mutagenic And Spectroscopic Investigation Of Ph Dependent Cooa Dna Binding, Brian R. Weaver

Chemistry Honors Papers

The carbon monoxide (CO) sensing heme protein, CooA, is a transcription factor which exists in several bacteria that utilize CO as an energy source. CooA positively regulates the expression of coo genes in the presence of CO such that the corresponding proteins may metabolize CO. The present studies have yielded the unexpected result that Fe(III) CooA binds DNA tightly at pH < 7, deviating from all previously reported work which indicate that CooA DNA binding is initiated only when the exogenous CO effector reacts with the Fe(II) CooA heme. This observation suggests that the disruption of one or more salt bridges upon effector binding may be a critical feature of the normal CooA activation mechanism. To test this possibility, several protein variants that eliminated a selected salt bridge for the CooA homolog from Rhodospirillum rubrum were prepared via site-directed mutagenesis. Samples of these variant proteins, which were overexpressed in Escherichia coli, were then characterized by spectroscopic methods and functional assays to investigate the impact these mutations had on CooA heme coordination …

Increasing Renewable Oil Content And Utility, William Richard Serson

Theses and Dissertations--Plant and Soil Sciences

Since the dawn of agriculture man has been genetically modifying crop plants to increase yield, quality and utility. In addition to selective breeding and hybridization we can utilize mutant populations and biotechnology to have greater control over crop plant modification than ever before. Increasing the production of plant oils such as soybean oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. We show that by manipulating a highly active acyl-CoA: diacylglycerol acyltransferase (DGAT) the hydrocarbon flux to oil in …

Dissecting The Histone-Binding Mechanism Of A Phd Finger Subtype, Daniel Boamah

Electronic Theses and Dissertations

Disordered tails of histones are critical information retrieval hub and thus, aberrations in the flow of information through these hubs are associated with a number of pathological consequences in human. Mechanism for retrieval of information from these hubs is achieved by protein-protein interaction, i.e. proteins dock onto histone tails to initiate chromatin signaling. Eukaryotes have a number of small peptide binding domains that have evolved to specifically interact with histone tails, and these domains called histone readers as they read the information encoded on histone tails. Plant homeodomain (hereafter PHD) finger, a binucleated zinc finger, family is one such histone …