Genetics and Genomics Commons^™

Transcription Analysis Of The Chlorovirus Paramecium Bursaria Chlorella Virus-1, Giane M. Yanai

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

Paramecium bursaria chlorella virus (PBCV-1), a member of the family Phycodnaviridae, is a large dsDNA, plaque-forming virus that infects the unicellular green alga Chlorella NC64A. The 331 kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To follow global transcription during PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes (CDS) was constructed. Competitive hybridization experiments were conducted employing cDNAs from poly A-containing RNAs obtained from cells at seven time points after virus infection. The results led to the following conclusions: i) the PBCV-1 replication cycle is temporally programmed and regulated; ii) 360 …

Genetic Effect Of The Dwarfing Genes On Some Culm Characteristics Associatcd With Lodging Resistance In Bread Wheat, Md. Mahbub Hasan

Md. Mahbub Hasan

Due to the challenge of screening traits related to lodging resistance under natural field conditions, selection for lodging resistant varieties in wheat breeding programs is difficult. The identification of easily measurable culm anatomical traits related to lodging resistance would simplify the selection process. The present study was conducted to determine the effect of dwarfing genes on culm anatomical traits related to lodging resistance in our of basal internode 1. Field and laboratory study was conducted in Shahjalal University of Science and Technology, Sylhet, Bangladesh with eight wheat genotypes having Rhr1, Rht2 dwarfing genes in them and a local land race …

Drosophila Adult Eye Model To Teach Scanning Electron Microscopy In An Undergraduate Cell Biology Laboratory, Meghana Tare, Oorvashi Roy Puli, Sarah M. Oros, Amit Singh

Biology Faculty Publications

We have devised an undergraduate laboratory exercise to study tissue morphology using fruit fly, Drosophila melanogaster, as the model organism. Drosophila can be reared in a cost effective manner in a short period of time. This experiment was a part of the undergraduate curriculum of the cell biology laboratory course aimed to demonstrate the use of scanning electron microscopy (SEM) technique to study the morphology of adult eye of Drosophila. The adult eye of Drosophila is a compound eye, which comprises of 800 unit eyes, and serves as an excellent model for SEM studies. We used flies that …

Protection Against Mucosal Shiv Challenge By Peptide And Helper-Dependent Adenovirus Vaccines, Eric A. Weaver, Pramod N. Nehete, Bharti P. Nehete, Stephanie J. Buchl, Donna Palmer, David C. Montefiori, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helperdependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5- immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than in …

The Role Of Human Endogenous Retroviruses In Renal Cell Carcinoma, Michele D. Tisdale

Biological Sciences Theses & Dissertations

Human endogenous retroviruses make up approximately 8-9% of the human genome. A number of expressed HERVs, those that are actively transcribing, have been associated with various cancers. Suppression mechanisms that control HERV expression often fail or become more permissive in tissues where expression should be restricted. Previous studies have identified HERV expression in breast cancer tissues, whereas normal tissue HERV expression remained suppressed. In addition, studies of DNA hypermethylation have correlated with the ability to contribute to cancer development. Hypermethylation of several tumor suppressor genes occurs frequently in cancers and alterations in promoter regions could contribute to the development of …

Archaeal Populations In Biological Soil Crusts From Arid Lands In North America, Tanya Soule, Ian J. Anderson, Shannon L. Johnson, Scott T. Bates, Ferran Garcia-Pichel

Tanya Soule

Archaea are common and abundant members of biological soil crust communities across large-scale biogeographic provinces of arid North America. Regardless of microbial community development, archaeal populations averaged 2 × 107 16S rRNA gene copies per gram of soil, representing around 5% of the prokaryotic (total calculated bacterial and archaeal) numbers assessed by quantitative-PCR. In contrast, archaeal diversity, determined by denaturing gradient gel electrophoresis fingerprinting and clone libraries of 16S rRNA genes, was very restricted. Only six different phylotypes (all Crenarchaea) were detected, three of which were very dominant. Some phylotypes were widespread, while others were typical of Southern desert areas.

Tracking Insertion Mutants Within Libraries By Deep Sequencing And A Genome-Wide Screen For Haemophilus Genes Required In The Lung, Jeffrey D. Gawronski, Sandy M. S. Wong, Georgia Giannoukos, Doyle V. Ward, Brian J. Akerley

Brian J. Akerley

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. …

Microbial Nad Metabolism: Lessons From Comparative Genomics, Francesca Gazzaniga, Rebecca Stebbins, Sheila Z. Chang, Mark A. Mcpeek, Charles Brenner

Dartmouth Scholarship

NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life …

Attempts To Cultivate Bacteria From Deep Subsurface Aquifers And Mountaintop Plant Communities, Eric D. Hughes, J. C. Bruckner, Duane P. Moser

Undergraduate Research Opportunities Program (UROP)

In the late 1990s, the limits of life were pushed even further when microorganisms were discovered thriving 2.5 km below the surface of the Earth in deep South African gold mines. These very simple communities were dominated by a single species of bacteria from within the phylum, Firmicutes. Desulforudis audaxviator remains unique to a sizeable portion of the South African deep subsurface. At depths below 2.5km, it comprises well over 99% of all organisms present, which presents a unique circumstance in which the environment has provided a natural pure culture. From this naturally occurring pure culture, environmental genomics was applied …

The Role Of Rpoe In Stationary Phase Mutagenesis In Bacillus, Turquoise C. Alexander, Eduardo A. Robleto

Undergraduate Research Opportunities Program (UROP)

Stationary phase mutagenesis is a phenomenon whereby random mutations are generated in non-dividing cells. In order to understand how these mutations arise, we use Bacillus subtilis, a gram positive rod-shaped model organism. It is hypothesize that increased transcription promotes stationary phase mutagenesis in this organism. We therefore examined the role of rpoE, a gene that encodes RNA polymerase ! subunit and proposed to influence efficiency of transcription. To this end, we will first generate a strain bearing a deletion in the rpoE gene. In order to determine if this gene is important for mutagenesis, we will examine the accumulation of …

The Role Of Recn In Stationary Phase Mutagenesis In Bacillus Subtilis, Lauren E. Johnson, Katherine R. Ona, Eduardo A. Robleto, Ronald E. Yasbin

Undergraduate Research Opportunities Program (UROP)

Here, we examine mutagenic programs that are independent of growth, such aspects of the evolutionary process are novel and have been implicated in the formation of cancers in animal cells and the acquisition of antibiotic resistance in animal pathogens. Adaptive or stationary phase mutagenesis is a genetic program to in increase diversity in cells under conditions of stress whereby cells escape non-dividing conditions. Previous research has shown that recombination functions are required to generate mutations that promote growth in Escherichia coli cells starved for carbon. This project tests the hypothesis that recombination functions are required for the generation of mutations …

Exploring Diversity Of Nitrate Reducing Thermophiles In Nevada Hot Springs, Jenny Lam, Jeremy A. Dodsworth, Brian P. Hedlund

Undergraduate Research Opportunities Program (UROP)

High rates of denitrification have been measured in Nevada geothermal hot springs, but little is known about the thermophiles that contribute to this activity. We hypothesize that heterotrophic bacteria in the genus Thermus are the most important denitrifiers in the springs. Alternatively, other microorganisms including chemolithotrophs may also be important. To test these hypotheses, several different strategies will be used to try to enrich and isolate nitrate-reducing microorganisms. Isolates will be identified by 16S rRNA gene PCR and sequencing. Subsequently, representative isolates will be chosen for nitrate reductase gene (narG) sequencing and for studies on the kinetics of nitrate reduction …

Rapid Evolution Of Sex-Pheromone-Producing Enzyme In Drosophila, Troy R. Shirangi, Héloïse D. Dufour, Thomas M. Williams, Sean B. Carroll

Biology Faculty Publications

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We …

Adding Upstream Sequence And A Downstream Reporter To The Bile Acid Inducible Promoter Of Clostridium Scindens Vpi 12708, Bryan Patrick Mason

Masters Theses & Specialist Projects

Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model …

A Comparative Genomics Approach To Understanding The Biosynthesis Of The Sunscreen Scytonemin In Cyanobacteria, Tanya Soule, Kendra Palmer, Qunjie Gao, Ruth M. Potrafka, Valerie Stout, Ferran Garcia-Pichel

Tanya Soule

Background The extracellular sunscreen scytonemin is the most common and widespread indole-alkaloid among cyanobacteria. Previous research using the cyanobacterium Nostoc punctiforme ATCC 29133 revealed a unique 18-gene cluster (NpR1276 to NpR1259 in the N. punctiforme genome) involved in the biosynthesis of scytonemin. We provide further genomic characterization of these genes in N. punctiforme and extend it to homologous regions in other cyanobacteria.

Results Six putative genes in the scytonemin gene cluster (NpR1276 to NpR1271 in the N. punctiforme genome), with no previously known protein function and annotated in this study as scyA to scyF, are likely involved in the assembly …

Identification Of Synechococcus Sp. Iu 625 Phycocyanin Gene And Bioinformatic Analyses Of Cyanobacterial Phycocyanin., Tin-Chun Chu, Aline Oliveira, Arti Rana, Lee Lee

Tin-Chun Chu, Ph.D.

No abstract provided.

Gene Expression Patterns Associated With The Biosynthesis Of The Sunscreen Scytonemin In Nostoc Punctiforme Atcc 29133 In Response To Uva Radiation, Tanya Soule, Ferran Garcia-Pichel, Valerie Stout

Tanya Soule

Under exposure to UV radiation, some cyanobacteria synthesize sunscreen compounds. Scytonemin is a heterocyclic indole-alkaloid sunscreen, the synthesis of which is induced upon exposure to UVA (long-wavelength UV) radiation. We previously identified and characterized an 18-gene cluster associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133; we now report on the expression response of these genes to a step-up shift in UVA exposure. Using quantitative PCR on cDNAs from the N. punctiforme transcriptome and primers targeting each of the 18 genes in the cluster, we followed their differential expression in parallel subcultures incubated with and without UVA. All …

Localization And Expression Of The Aer Receptor In Escherichia Coli, Daniel Salcedo

Loma Linda University Electronic Theses, Dissertations & Projects

In Escherichia coli, chemotactic movement towards an energetically-favorable environment is mediated by five transmembrane chemoreceptors. These sensory proteins respond to numerous environmental signals, including amino acids, sugars, and pH. The aerotaxis receptor, Aer, is an intracellular sensor that responds to oxygen concentration and other parameters affecting cellular energy levels. Bacterial chemotaxis receptors form dimeric signaling units that organize into tetramers and hexamers and form large clusters at the cell poles. A cluster arrangement could increase receptor sensitivity by lateral communication between dimers, thus amplifying the signal from one dimer. We have shown that Aer forms dimers, trimer of dimers, …

Contribution Of Ledgf/P75 To Prostate Cancer Chemoresistance, Melanie Mediavilla-Varela

Loma Linda University Electronic Theses, Dissertations & Projects

Prostate cancer (PCa) is the second leading cause of male cancer death in the United States, and is considered a health disparity because of its disproportionate incidence and mortality in African American (AA) males. A major challenge in PCa treatment is its recurrence into hormone refractory prostate cancer (HRPC). Docetaxel is the standard of care for first line chemotherapy to HRPC. Unfortunately, most patients ultimately manifest resistance to DTX and succumb to the disease. Circumventing DTX resistance in HRPC will require better understanding of the mechanisms by which this drug kills prostate tumor cells, and the genes that promote this …

Altered Regulation Of Aquaporin Gene Expression In Allergen And Il-13-Induced Mouse Models Of Asthma, Carissa M. Krane, Bijia Deng, Venkateshwar Mutyam, Casey A. Mcdonald, Stephen Pazdziorko, Lawrence Mason, Samuel Goldman, Marion Kasaian, Divya Chaudhary, Cara Williams, Melisa W.Y. Ho

Biology Faculty Publications

IL-13 is known to affect many processes that contribute to an asthmatic phenotype, including inflammation, fibrosis, and mucus production. Members of the aquaporin (AQP) family of transmembrane water channels are targets of regulation in models of lung injury and inflammation. Therefore, we examined AQP mRNA and protein expression in allergen and IL-13-induced mouse models of asthma. Lungs from ovalbumin sensitized and ovalbumin challenged (OVA/OVA) and IL-13 treated mice showed airway thickening, increased mucus production, and pulmonary eosinophilia. Pulmonary function tests showed a significant increase in methacholine-induced airway hyperreactivity in OVA/OVA and IL-13-treated mice as compared with controls. Quantitative PCR analysis …

Comparison Of Replication-Competent, First Generation, And Helper-Dependent Adenoviral Vaccines, Eric A. Weaver, Pramod N. Nehete, Stephanie S. Buchl, Julien S. Senac, Donna Palmer, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and …

Development Of A Gene Silencing Dna Vector Derived From A Broad Host Range Geminivirus, Edward M. Golenberg, D Noah Sather, Leandria C. Hancock, Kenneth J. Buckley, Natalie M. Villafranco, David M. Bisaro

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.

Results

The construction of a gene silencing vector derived from the …

High Resolution Single Molecule Optical Localization Of Multiple Fluorophores On Dna Origami Constructs Fluorophores On Dna Origami Constructs, Anuradha Rajulapati

Theses, Dissertations and Capstones

An ongoing challenge in the development of nanoelectronics and nanophotonics is the nondestructive, high-resolution localization in space of single molecules and multi-molecular assemblies. The apparent barrier to the use of optical microscopy at the sub-100 nm scale is the well known Abbe Limit, the diffraction limit to resolution. This laboratory has adapted a technique called Single-molecule high resolution imaging with photobleaching (SHRIMP). We have developed methods for utilizing SHRIMP for the determination of the separation of two fluorophores in single DNA origami constructs.

DNA Origami is extremely useful because it can address nanocomponents down to 2nm separation. It should be …

Patterning Defects In Silkworm Embryos Analysed Through Cuticle Preparations, Amit Singh, Madhuri Kango-Singh, K. P. Gopinathan

Biology Faculty Publications

The mulberry silkworm, Bombyx mori, a holometabolous lepidopteran insect, has a metameric body plan. Due to its functional adaptation, B. mori presents some unique deviations in its pal/ern from the evolutionarily advanced dipteran insect, Drosophila melanogaster. Previous studies on mutant phenotype analysis in B. mori have been carried out in late stages of larval development. Here we employ, the cuticle preparation approach during embryonic development to study morphological landmarks associated with B. mori, Eri, another race a/silkworm, and pattern defects associated with Ekp mutant of B.mori. The homeotic mutant Ekp, generates ectopic abdominallegs, a feature …

Intradermal Delivery Of Plasmid Vegf(165) By Electroporation Promotes Wound Healing, Bernadette Ferraro, Yolmari Cruz, Domenico Coppola, Richard Heller

Bioelectrics Publications

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF₁₆₅ (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal …

Electroporation For The Delivery Of Dna-Based Vaccines And Immunotherapeutics: Current Clinical Developments, Angela M. Bodles-Brakhop, Richard Heller, Ruxandra Draghia-Akli

Bioelectrics Publications

Electroporation (EP) has been used in basic research for the past 25 years to aid in the transfer of DNA into cells in vitro. EP in vivo enhances transfer of DNA vaccines and therapeutic plasmids to the skin, muscle, tumors, and other tissues resulting in high levels of expression, often with serological and clinical benefits. the recent interest in nonviral gene transfer as treatment options for a vast array of conditions has resulted in the refinement and optimization of EP technology. current research has revealed that EP can be successfully used in many species, including humans. clinical trials are …

An Algorithm For Identifying Novel Targets Of Transcription Factor Families: Application To Hypoxia-Inducible Factor 1 Targets, Yue Jiang, Bojan Cukic, Donald A. Adjeroh, Heath D. Skinner, Jie Lin, Qingxi J. Shen, Bing-Hua Jiang

Electrical & Computer Engineering Faculty Research

Efficient and effective analysis of the growing genomic databases requires the development of adequate computational tools. We introduce a fast method based on the suffix tree data structure for predicting novel targets of hypoxia-inducible factor 1 (HIF-1) from huge genome databases. The suffix tree data structure has two powerful applications here: one is to extract unknown patterns from multiple strings/sequences in linear time; the other is to search multiple strings/sequences using multiple patterns in linear time. Using 15 known HIF-1 target gene sequences as a training set, we extracted 105 common patterns that all occur in the 15 training genes …

Compensatory Evolution Of Gene Regulation In Response To Stress By Escherichia Coli Lacking Rpos, Daniel M. Stoebel, Karsten Hokamp, Michael S. Last, Charles J. Dorman

All HMC Faculty Publications and Research

The RpoS sigma factor protein of Escherichia coli RNA polymerase is the master transcriptional regulator of physiological responses to a variety of stresses. This stress response comes at the expense of scavenging for scarce resources, causing a trade-off between stress tolerance and nutrient acquisition. This trade-off favors non-functional rpoS alleles in nutrient-poor environments. We used experimental evolution to explore how natural selection modifies the regulatory network of strains lacking RpoS when they evolve in an osmotically stressful environment. We found that strains lacking RpoS adapt less variably, in terms of both fitness increase and changes in patterns of transcription, than …

Protection Against Mucosal Shiv Challenge By Peptide And Helper-Dependent Adenovirus Vaccines, Eric A. Weaver, Pramod N. Nehete, Bharti P. Nehete, Stephanie J. Buchl, Donna Palmer, David C. Montefiori, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helper- dependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5- immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than …

Comparison Of Replication-Competent, First Generation, And Helper-Dependent Adenoviral Vaccines, Eric A. Weaver, Pramod N. Nehete, Stephanie S. Buchl, Julien S. Senac, Donna Palmer, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and …