Molecular Genetics Commons^™

Analysis Of The Clear Plaque Phenotype Of The Bacteriophage Hk75, Phani Chandrika Kunapuli

Masters Theses & Specialist Projects

The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its …

Characterizing And Treating The Neuropathology Of Tuberous Sclerosis Complex In The Mouse, Sharon W. Way

Dissertations and Theses (Open Access)

Tuberous sclerosis complex (TSC) is a multisystem, autosomal dominant disorder affecting approximately 1 in 6000 births. Developmental brain abnormalities cause substantial morbidity and mortality and often lead to neurological disease including epilepsy, cognitive disabilities, and autism. TSC is caused by inactivating mutations in either TSC1 or TSC2, whose protein products are known inhibitors of mTORC1, an important kinase regulating translation and cell growth. Nonetheless, neither the pathophysiology of the neurological manifestations of TSC nor the extent of mTORC1 involvement in the development of these lesions is known. Murine models would greatly advance the study of this debilitating disorder. This thesis …

Rmi1 Is Essential For Early Embryonic Development And Attenuation Of Tumor Development, Haoyi Chen

Dissertations and Theses (Open Access)

RMI1 (BLM-Associated Protein 75 or Blap75) is highly conserved from yeast to human. Previous studies have shown that hRMI1 is required for BLM/TopoIIIα/RMI1 complex stability and function. However, in vivo functions of RMI1 remain elusive. To address this question, I generated RMI1 knockout mice by homologous replacement targeting. While RMI1+/- mice showed no obvious phenotype, deletion of both RMI1 alleles leads to early embryonic lethality before implantation. I then generated RMI1/p53 double knockout mice. After ionizing radiation treatment at 4Gy, RMI1/p53 double-heterzygous mice showed shortened tumor latency and aggressive tumor types when comparing with wild type, RMI1+/- and p53+/- control …

Characterization Of The Agrobacterium Tumefaciens Virb2 Pilin Of The Virb/D4 Type Iv Secretion System, Jennifer Kerr

Dissertations and Theses (Open Access)

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS …

Agrobacterium Virb10 Contributions To Type Iv Substrate Secretion, T-Pilus Biogenesis, And Outer Membrane Pore Formation, Isaac Garza

Dissertations and Theses (Open Access)

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) …

Complement Factor H-Related Proteins Cfhr2 And Cfhr5 Represent Novel Ligands For The Infection-Associated Crasp Proteins Of Borrelia Burgdorferi, Corinna Siegel, Teresia Hallström, Christine Skerka, Hannes Eberhardt, Barbara Uzonyi, Tobias Beckhaus, Michael Karas, Reinhard Wallich, Brian Stevenson, Peter F. Zipfel, Peter Kraiczy

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi.

METHODOLOGY/PRINCIPAL FINDINGS: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins …

Dorsal Eye Selector Pannier (Pnr) Suppresses The Eye Fate To Define Dorsal Margin Of The Drosophila Eye, Sarah M. Oros, Meghana Tare, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Axial patterning is crucial for organogenesis. During Drosophila eye development, dorso-ventral (DV) axis determination is the first lineage restriction event. The eye primordium begins with a default ventral fate, on which the dorsal eye fate is established by expression of the GATA-1 transcription factor pannier (pnr). Earlier, it was suggested that loss of pnr function induces enlargement in the dorsal eye due to ectopic equator formation. Interestingly, we found that in addition to regulating DV patterning, pnr suppresses the eye fate by downregulating the core retinal determination genes eyes absent (eya), sine oculis (so) and dacshund (dac) to define the …

Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication Via Interaction With Ddx3x, Chaomin Sun, Cara T. Pager, Guangxiang Luo, Peter Sarnow, Jamie H. D. Cate

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16-36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent …

Sialic Acid Transport And Catabolism Are Cooperatively Regulated By Siar And Crp In Nontypeable Haemophilus Influenzae, Jason W. Johnston, Haider Shamsulddin, Anne-Frances Miller, Michael A. Apicella

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: The transport and catabolism of sialic acid, a critical virulence factor for nontypeable Haemophilus influenzae, is regulated by two transcription factors, SiaR and CRP.

RESULTS: Using a mutagenesis approach, glucosamine-6-phosphate (GlcN-6P) was identified as a co-activator for SiaR. Evidence for the cooperative regulation of both the sialic acid catabolic and transport operons suggested that cooperativity between SiaR and CRP is required for regulation. cAMP was unable to influence the expression of the catabolic operon in the absence of SiaR but was able to induce catabolic operon expression when both SiaR and GlcN-6P were present. Alteration of helical phasing supported …

Association Between Chronic Liver And Colon Inflammation During The Development Of Murine Syngeneic Graft-Versus-Host Disease, Jason Anthony Brandon, Jacqueline Perez-Rodriguez, C. Darrell Jennings, Donald A. Cohen, Vishal J. Sindhava, Subbarao Bondada, Alan M. Kaplan, J. Scott Bryson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The murine model of cyclosporine A (CsA)-induced syngeneic graft-versus-host disease (SGVHD) is a bone marrow (BM) transplantation model that develops chronic colon inflammation identical to other murine models of CD4⁺ T cell-mediated colitis. Interestingly, SGVHD animals develop chronic liver lesions that are similar to the early peribiliary inflammatory stages of clinical chronic liver disease, which is frequently associated with inflammatory bowel disease (IBD). Therefore, studies were initiated to investigate the chronic liver inflammation that develops in the SGVHD model. To induce SGVHD, mice were lethally irradiated, reconstituted with syngeneic BM, and treated with CsA. All of the SGVHD animals …

Bpab, A Novel Protein Encoded By The Lyme Disease Spirochete's Cp32 Prophages, Binds To Erp Operator 2 Dna, Logan H. Burns, Claire A. Adams, Sean P. Riley, Brandon L. Jutras, Amy Bowman, Alicia M. Chenail, Anne E. Cooley, Laura A. Haselhorst, Alisha M. Moore, Kelly Babb, Michael G. Fried, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5′ of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA …

Splice Factor Sfrs6: Regulation By P53 And Effect On Il-24 Splicing, Erin L. Schmidt

Loma Linda University Electronic Theses, Dissertations & Projects

Alternative splicing of RNA transcripts is emerging as a major mechanism for expanding the proteome. Splice pattern changes are found in numerous diseases and cancers, indicating the importance of tight control over this process. While the mechanism of splicing has been described in detail, it is less clear how a cell is able to modify its splicing patterns in response to different conditions. Our lab previously demonstrated that DNA damage can induce SFRS6, the gene coding for splicing factor SRp55, thereby leading to changes in the splicing patterns of several target genes related to survival and apoptosis. We also …

Cross-Reactivity Of Antibodies Against Leptospiral Recurrent Uveitis-Associated Proteins A And B (Lrua And Lrub) With Eye Proteins, Ashutosh Verma, Pawan Kumar, Kelly Babb, John F. Timoney, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to …

The Domains Of The Catalytic Subunit Of The Eukaryotic Rna Degrading Exosome, Rrp44p, Have Distinct Functions, Daneen Schaeffer

Dissertations and Theses (Open Access)

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA.

The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, …

E2f1 And Tumor Suppression: The Role Of P21, Mirnas, And The Dna Damage Response, Regina L. Weaks

Dissertations and Theses (Open Access)

E2F1 is a multi-faceted protein that has roles in a number of important cellular processes including cell cycle regulation, apoptosis, proliferation, and the DNA damage response (DDR). Moreover, E2F1 has opposing roles in tumor development, acting as either a tumor suppressor or an oncogene depending on the context. In human cancer, E2F1 is often deregulated through aberrations in the Rb-p16INK4a-cyclin D1 pathway. In these studies we examined three mechanisms by which E2F1 might mediate its tumor suppressive properties: p21-induced senescence, miRNAs, and the DNA damage response. We found that E2F1 acts as a tumor suppressor in response to ras activation …

Regulation Of Morphogenesis In Filamentous Fungi, Haoyu Si

School of Biological Sciences: Dissertations, Theses, and Student Research

One of the distinguishing features of fungal cells is their highly polarized model of growth. Both yeast cells and hyphal cells grow by cell surface expansion at specified cortical sites. Although the same general mechanisms are likely to be involved in controlling the establishment of hyphal polarity in budding yeast and filamentous fungi, it is noticeable that hyphal cells are organized in a fundamentally different manner to yeast dells. For example, hyphal cells organize formins, septins and actins at the division site while simultaneously retain the same machinery at the tip; whereas yeast cells undergo a transient period of isotropic …

Determining The Fate Of Hybridized Genomes In The Allopolyploid Brassica Napus, Tina Y. Wang

Master's Theses

Polyploidy is widely acknowledged as a widespread mechanism in the evolution and speciation of the majority of flowering plants. Allopolyploid forms through interspecific hybridization and whole genome duplication. While allopolyploids may display increased vigor relative to their progenitors, they can also face challenges to fertility following hybridization. Genetic changes in allopolyploids result from recombination between the hybridized subgenomes, which can influence phenotype and ultimately determine fitness of future generations. To study dynamic changes that follow allopolyploid formation, Brassica napus lineages were derived by hybridizing Brassica oleracea and Brassica rapa. Two lineages of B. napus were analyzed for genetic and phenotypic …

Fetal Programming By Nicotine Increases Cardiac Susceptibility To Ischemic Injury, Jennifer Charlotte Alexie Lawrence

Loma Linda University Electronic Theses, Dissertations & Projects

Fetal programming is the area of study that focuses on the prenatal origins of adult onset disorders. Previous studies have associated an adverse prenatal environment with the onset of physiologic and metabolic diseases during adulthood. Fetal malnutrition, hypoxia, and exposure to drugs - such as cocaine and nicotine - have been associated with adult disease states. Cigarette smoking is the number one cause of preventable death in the developed world. Among the many dangerous chemicals found in tobacco products is nicotine, the compound responsible for the addictive nature of tobacco use. Nicotine use during pregnancy is a known cause of …

Regulation Of Pim1 Under Hypoxia In Prostate Cancer, Eva Sahakian

Loma Linda University Electronic Theses, Dissertations & Projects

A defining characteristic of solid tumors is the capacity to divide and spread under conditions of nutrient deprivation and limited oxygen availability. These microenvironmental stresses arise from structural abnormalities in tumor vessels that lead to aberrant microcirculation. Hypoxia acts as a physiological “selection pressure” in the progression of cancer by activating pathways and enhancing the expression of specific genes in tumor cells which eventually diminish their apoptotic potential. Ultimately, hypoxic microenvironment functions as a “stress factor”, selecting cells with the ability to survive and divide under anoxic conditions. The members of the PIM family of cytoplasmic serine threonine kinases are …

Development Of Representative Species-Level Molecular Markers And Morphological Character Analysis Of Leucothoid Amphipods (Crustacea: Amphipoda), Kristine Nicolle White

Dissertations

Leucothoid amphipods were investigated using morphology and molecular rDNA gene sequence fragments. The morphological diagnostic characters for traditional taxonomy have been clarified, a molecular marker for representative species has been developed, and one of the current anamorph-leucomorph connections has been confirmed with molecular sequence data. Ultimately this study has combined traditional morphological and modern molecular methods to clarify the taxonomy and to propose a preliminary phylogeny of the Leucothoidae. Analysis of 18S rDNA gene fragments from 13 species in two genera supported the current morphological species designations and the separation of the family into two clades. Combined analysis of 18S …

Molecular Analysis Of Trypanosoma Cruzi Isolates Obtained From Raccoons (Procyon Lotor) In Warren And Barren Counties Of Kentucky, Lipeng Bi

Masters Theses & Specialist Projects

Trypanosoma cruzi, the etiologic agent of Chagas disease, infects a variety of wild mammals in the southern United States, but it has only recently been isolated from raccoons trapped in the state of Kentucky. The purpose of the present study was to use a molecular genotyping approach, followed by DNA sequencing to determine the genotypes (type I, or types IIa-IIe) of 15 of the Kentucky isolates. DNA samples were prepared from 15 T. cruzi- isolates using a Qiagen mini kit, and PCR amplification was performed using published primers for the 24S α rDNA sequence (D71 and D72), the …

Xenobiotic Metabolism Genes And Clubfoot, Amy M. Sommer

Dissertations and Theses (Open Access)

Idiopathic or isolated clubfoot is a common orthopedic birth defect that affects approximately 135,000 children worldwide. It is characterized by equinus, varus and adductus deformities of the ankle and foot. Correction of clubfoot involves months of serial manipulations, castings and bracing, with surgical correction needed in forty percent of cases. Multifactorial etiology has been suggested in numerous studies with both environmental and genetic factors playing an etiologic role. Maternal smoking during pregnancy is the only common environmental factor that has consistently been shown to increase the risk for clubfoot. Moreover, a positive family history of clubfoot and maternal smoking increases …

Validation Of The Activation Of Aurora B Kinase By Caenorhabditis Elegans To Usled-Like Kinase And The Identification Of Cyclin B3 As A Phospho-Specific Tlk-1 Interactor, Gary Michael Deyter

Dissertations and Theses (Open Access)

A hallmark of tumorigenesis and certain birth defect syndromes is the loss of ploidy that can result from incorrect chromosome segregation. Chromosomes that are not partitioned properly during mitosis are often fragmented, changing the genetic makeup of daughter cells. Inheriting extrachromosomal fragments that contain cell survival genes or losing chromosomal loci that encode tumor suppressors can promote tumor development. Thus, it is essential to elucidate molecular mechanisms required for correct chromosome segregation. Chromosomes are connected to mitotic spindle microtubules by way of a proteinacous, chromosome-bound organelle called the kinetochore. Two decades of research have confirmed that the conserved Aurora B/AIR-2 …

Analysis Of Variation In Clubfoot Candidate Genes, Audrey R. Ester

Dissertations and Theses (Open Access)

Isolated clubfoot, a common birth defect occurring in more than 135,000 livebirths worldwide each year, is associated with significant health care and financial burdens. Clubfoot is defined by forefoot adduction, hindfoot varus, midfoot cavus and hindfoot equinus. Isolated clubfoot, which is the focus of these studies, is distinct from syndromic clubfoot because there are no other associated malformations. Population, family, twin and segregation analysis studies provide evidence that genetic and environmental factors play an etiologic role in isolated clubfoot. The studies described in this thesis were performed to define the role of genetic variation in isolated clubfoot. Interrogation of a …

The Role Of Ecdysone Signaling In Fat-Body Tissue Remodeling And Pupal Metabolism, Nichole Dinell Bond

UNLV Theses, Dissertations, Professional Papers, and Capstones

Holometabolous insects undergo an astonishing transition during their development. During metamorphosis, the larva dramatically changes form and becomes an adult fly. During this process obsolete larval tissues must be eliminated, while tissues required for further development are retained and often remodeled to meet the needs of the adult fly. Tissue remodeling is characterized by morphological changes of the cells in a tissue mass. In many cases, remodeling is characterized by dissociation of the tissue mass, releasing cells to move freely around the body cavity. This process is also common in wound healing and is a key feature of human disease …

Molecular Analysis Of B3galtl In A Patient With Peters' Plus Syndrome, Taiyabah Naeem

Undergraduate University Honors Capstones

Chromosomal rearrangement such as microdeletions or microduplications, which can alter the dosage of one or more genes, are commonly seen in patients with multiple congenital anomalies (MCA). Alternatively, the discovery of a microdeletion may sometimes uncover an autosomal recessive disorder if the gene on the non-deleted allele carries a mutation. This study reports molecular analysis of a patient with MCAs, including an enlarged head, developmental delay, clouding of the corneas, and short stature. Peters Plus Syndrome (PPS) is characterized as an autosomal recessive disorder. Phenotypical symptoms include various eye abnormalities and structural organ defects. The B3GGALTL has been found to …

The Rho Family Gtpase: Determining Gef Specificity Through Recombinant Expression, Farrah Steinke

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Quasispecies-Like Behavior Observed In Catalytic Rna Populations Evolving In A Test Tube, Carolina Diaz Arenas, Niles Lehman

Chemistry Faculty Publications and Presentations

Background: During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous.

Results: Using continuous in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen …

Structural Dynamics And Signaling Roles Of The Aer Pas And F1 Regions, Asharie J. Campbell

Loma Linda University Electronic Theses, Dissertations & Projects

The Aer aerotaxis receptor in Escherichia coli mounts such a rapid response to redox that E. coli changes its swimming behavior within 100 ms after an oxygen pulse. This receptor is a membrane-bound homodimer, and it monitors internal energy (redox) via an FAD cofactor bound to a cytoplasmic N-terminal PAS domain. Understanding PAS sensing is important, as PAS domains comprise a superfamily of more than 25,000 members from all kingdoms. This study focused on the Aer N-terminal PAS sensor, as well as the region tethering it to the membrane anchor, known as the F1. Previous genetic studies on these regions …

Proteomic Analysis Of Iron Acquisition, Metabolic And Regulatory Responses Of Yersinia Pestis To Iron Starvation, Rembert Pieper, Shih-Ting Huang, Prashanth P. Parmar, David J. Clark, Hamid Alami, Robert D. Fleischmann, Robert D. Perry, Scott N. Peterson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

BACKGROUND: The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26°C and 37°C).

RESULTS: Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis …