Browsing by Subject "Mutagenesis"

Now showing 1 - 10 of 15
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    A systemic approach to identify non-abundant immunogenic proteins in Lyme disease pathogens
    (American Society for Microbiology, 2024) Yaş, Ozlem Buyuktanir; Coleman, Adam S.; Lipman, Rachel M.; Sharma, Kavita; Raghunandanan, Sajith; Alanazi, Fuad; Rana, Vipin S.; Kitsou, Chrysoula; Yang, Xiuli; Pal, Utpal; Microbiology and Immunology, School of Medicine
    Borrelia burgdorferi, the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as OspABC- mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the OspABC- mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection. IMPORTANCE: The present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease.
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    Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
    (2012-09-27) Whybrew, Jennafer Marie; Bard, Martin; Lees, N. Douglas; Blacklock, Brenda
    Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.
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    Analysis of integration sites of transgenic sheep generated by lentiviral vectors using next-generation sequencing technology
    (2014-07-31) Chen, Yu-Hsiang; Malkova, Anna; Cornetta, Kenneth; Randall, Stephen Karl, 1953-; Atkinson, Simon
    The development of new methods to carry out gene transfer has many benefits to several fields, such as gene therapy, agriculture and animal health. The newly established lentiviral vector systems further increase the efficiency of gene transfer dramatically. Some studies have shown that lentiviral vector systems enhance efficiency over 10-fold higher than traditional pronuclear injection. However, the timing for lentiviral vector integration to occur remains unclear. Integrating in different stages of embryogenesis might lead to different integration patterns between tissues. Moreover, in our previous study we found that the vector copy number in transgenic sheep varied, some having one or more copies per cells while other animals having less than one copy per cell suggesting mosaicism. Here I hypothesized that injection of a lentiviral vector into a single cell embryo can lead to integration very early in embryogenesis but can also occur after several cell divisions. In this study, we focus on investigating integration sites in tissues developing from different germ layers as well as extraembryonic tissues to determine when integration occurs. In addition, we are also interested in insertional mutagenesis caused by viral sequence integration in or near gene regions. We utilize linear amplification-mediated polymerase chain reaction (LAM-PCR) and next- generation sequencing (NGS) technology to determine possible integration sites. In this study, we found the evidence based on a series of experiments to support my hypothesis, suggesting that integration event also happens after several cell divisions. For insertional mutagenesis analysis, the closest genes can be found according to integration sites, but they are likely too far away from the integration sites to be influenced. A well-annotated sheep genome database is needed for insertional mutagenesis analysis.
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    Biochemical applications of DsRed-monomer utilizing fluorescence and metal-binding affinity
    (2011-03-09) Goulding, Ann Marie; Deo, Sapna K.; Oh, Kyungsoo; Davidson, Amy; Simpson, Garth
    The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.
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    Break-Induced Replication is a Source of Mutation Clusters Underlying Kataegis
    (Elsevier B.V., 2014-06) Sakofsky, Cynthia J.; Roberts, Steven A.; Malc, Ewa; Mieczkowski, Piotr A.; Resnick, Michael A.; Gordenin, Dmitry A.; Malkova, Anna; Department of Biology, School of Science
    Clusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within long single-strand (ss) DNA formed at resected double-strand breaks and dysfunctional replication forks. We identify here double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination and strand bias of the mutation spectrum was consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Together, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species.
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    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, Lei
    Fatty 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.
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    Generation of conditional mutants to dissect essential gene fuction in chlamydia trachomatis
    (2016-12-07) Brothwell, Julie Ann; Nelson, David E.; Bauer, Margaret E.; Gilk, Stacey D.; Sullivan, William J., Jr.
    Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease. Chlamydia spp. are all obligate intracellular organisms that undergo a biphasic developmental cycle within a vacuole termed the inclusion. Infectious, non metabolically active elementary bodies (EBs) are endocytosed and differentiate into non infectious, metabolically active reticulate bodies (RBs) before re-differentiating back into EBs. The chlamydial factors that mediate these differentiation events are mostly unknown. Comparative genomics revealed that Chlamydia spp. have small, highly conserved genomes, suggesting that many of their genes may be essential. Genetic manipulation strategies for Chlamydia spp. are in their infancy, and most of these cannot be used to inactivate essential genes. We generated a clonal ethyl methanesulfonate (EMS)-mutagenized C. trachomatis library and screened it for temperature sensitive (TS) mutants that produced fewer inclusions at either 32°C or 40°C compared to 37°C. Because EMS mutagenesis elicited multiple mutations in most of the library isolates, we also developed a novel lateral gene transfer strategy for mapping mutations linked to TS phenotypes. We identified TS alleles of genes that are essential in other bacteria and that are involved in diverse biological processes including DNA replication, protein synthesis, carbohydrate metabolism, fatty acid biosynthesis, and energy generation, as well as in highly conserved chlamydial hypothetical genes. TS DNA polymerase (dnaEts) and glutamyl-tRNA synthestase (gltXts) mutants were characterized further. Both the dnaEts and gltXts mutants failed to replicate their genomes at 40°C but exhibited unique signs of stress. Chlamydial DNA replication begins by 12 hpi and protein synthesis begins by 2 hpi. However, inclusion expansion and replication of both of the mutants could be rescued by shifting to them to 37°C prior to mid-late development. Since gltXts is likely unable to produce aminoacyl-tRNAs at 40°C, our observation suggests that de novo chlamydial translation uses a pre-existing pool of aminoacyl-tRNA in EBs. Genetic suppressor analysis indicated that the inability of the dnaEts mutant to replicate its genome at 40°C might be linked to an inability of mutant DnaE to bind the DNA template. The tools and mutants we have identified will be invaluable assets for investigating many essential aspects of chlamydial biology.
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    The influence of the Ku80 carboxy-terminus on activation of the DNA-dependent protein kinase and DNA repair is dependent on the structure of DNA cofactors
    (2013-11) Woods, Derek S.; Turchi, John J.; Harrington, Maureen A.; Malkova, Anna L.; Takagi, Yuichiro
    In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.
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    Loss of photoreversibility in E. coli B/r
    (1981) Kristoff, Steve