Genetics Commons^™

Dna Polymerase Θ (Polq) And The Cellular Defense Against Dna Damage, Matthew J. Yousefzadeh

Dissertations and Theses (Open Access)

In mammalian cells, DNA polymerase θ (POLQ) is an unusual specialized DNA polymerase whose in vivo function is under active investigation. The protein is comprised of an N-terminal helicase-like domain, a C-terminal DNA polymerase domain, and a large central domain that spans between the two. This arrangement is also found in the Drosophila Mus308 protein, which helps confer resistance to DNA interstrand crosslinking agents. Homologs of POLQ and Mus308 are found in eukaryotes, including plants, but a comparison of phenotypes suggests that not all of these genes are functional orthologs. Flies with defective Mus308 are sensitive to DNA interstrand crosslinking …

Repsa-Directed Identification Of Dna-Binding Specificity For Orphan Transcription Factors, Kamir Hiam

Symposium of Student Scholars

The function of many genes and the biological roles of their encoded products are still not well characterized. Given the sequence-specific DNA-binding properties of transcription factor proteins, it is possible to purify them, identify the responsible polypeptide(s), determine their consensus binding sequences, and identify their genomic binding sites. Thus, one can go from cellular extract to proposed biological regulatory roles in relatively short order. Our goal is to identify and characterize orphan DNA-binding proteins in the model organism E. coli K12 using the novel combinatorial technique, REPSA (Restriction Endonuclease Protection Selection Amplification), as well as further develop the REPSA to …

Investigating The Initial Detection Stage Of Meiotic Silencing By Unpaired Dna In The Model Organism Neurospora Crassa, Pegan A. Sauls

Theses and Dissertations

In an attempt to neutralize transposable elements or retrovirus invasions Neurospora crassa will rely on one of its many genome defense mechanism, Meiotic Silencing by Unpaired DNA (MSUD). MSUD works in a two-step process that first detects unpaired sequences between homologous chromosomes followed by downstream silenced expression of the sequence. The ultimate silencing stage of MSUD is widely accepted to operate through an RNAi-like system. However, the mechanics of the detection step of MSUD remains elusive. The research presented attempts to elaborate on how the initial stage of MSUD occurs and its specifics. First, a genetic approach is utilized to …

Primer Efficacy In The Dna Barcoding Of Spiders, Rhennetta Jo Bork

Honors Program Theses

DNA barcoding is the process of amplifying a 650 base pair segment of the sequence of the mitochondrial gene cytochrome c oxidase (COI), and amplifying this gene with a polymerase chain reaction (PCR). It is used to help identify and distinguish animal species and also to help determine genetic differences in species. DNA barcoding can be especially useful when working with spiders since they tend to be very small and hard to distinguish. However, achieving a DNA barcode can be difficult and thus techniques to improve the method of DNA barcoding can be helpful. This research looked into the different …

An Active Role For The Ribosome In Determining The Fate Of Oxidized Mrna, Carrie L. Simms, Benjamin H. Hudson, John W. Mosior, Ali S. Rangwala, Hani S. Zaher

Biology Faculty Research

Chemical damage to RNA affects its functional properties and thus may pose a significant hurdle to the translational apparatus; however, the effects of damaged mRNA on the speed and accuracy of the decoding process and their interplay with quality-control processes are not known. Here, we systematically explore the effects of oxidative damage on the decoding process using a well-defined bacterial in vitro translation system. We find that the oxidative lesion 8-oxoguanosine (8-oxoG) reduces the rate of peptide-bond formation by more than three orders of magnitude independent of its position within the codon. Interestingly, 8-oxoG had little effect on the fidelity …

A Homogenizing Process Of Selection Has Maintained An 'Ultra-Slow' Acetylation Nat2 Variant In Humans, Blandine Patillon, Pierre Luisi, Estella S. Poloni, Sotiria Boukouvala, Pierre Darlu, E. Genin, Audrey Sabbagh

Human Biology Open Access Pre-Prints

N-acetyltransferase 2 (NAT2) is an important enzyme involved in the metabolism of a wide spectrum of naturally occurring xenobiotics, including therapeutic drugs and common environmental carcinogens. Extensive polymorphism in NAT2 gives rise to a wide interindividual variation in acetylation capacity which influences individual susceptibility to various drug-induced adverse reactions and cancers. Striking patterns of geographic differentiation have been described for the main slow acetylation variants of the NAT2 gene, suggesting the action of natural selection at this locus. In the present study, we took advantage of the whole-genome sequence data available from the 1000 Genomes project to investigate the …

Key Residues Of Human Cytoplasmic Protein Tyrosine Phosphatase-A And -B For Substrate Binding And Specificity, Byunghyun Park

Open Access Theses

Reversible tyrosine phosphorylation plays an important role in signaling pathways that are essential for regulating cellular growth, differentiation and metabolism. Moreover, several human diseases such as diabetes, obesity and cancers are associated with the deregulation of protein tyrosine phosphatases (PTPs). Several studies provide evidence that PTPs not only contribute to cellular differentiation, but over-expression of these molecules also leads to transformation of non-transfomed cells as well. Based on these results, designing specific PTP inhibitors may ultimately function as potential therapeutic agents to treat various diseases including cancer, diabetes, and autoimmune diseases. EphA2 is a receptor tyrosine kinase which is hypo-phosphorylated …

Clpxp Modulates Cell Growth And Morphology In Cell Shape Mutants Of E.Coli, Ryann Murphy

Senior Honors Projects

ClpXP modulates cell growth and morphology in cell shape mutants of E. coli

Ryann Murphy¹ and Jodi L. Camberg¹

¹University of Rhode Island, Department of Cell and Molecular Biology, Kingston, RI, 02881

Penicillin Binding Proteins (PBPs) are a family of prokaryotic membrane proteins named for their propensity to bind the antibiotic penicillin and are involved in remodeling and deposition of peptidoglycan. In wild type Escherichia coli cells, the uniform rod shape is conserved across generations. E.coli cells containing multiple deletions of Low Molecular Weight (LMW) PBPs exhibit irregular shapes. LMW PBP5 (dacA) is a potential …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

University Scholar Projects

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Modeling The Adaptive Immune Response To Mutation-Generated Antigens, Rory J. Geyer

Honors Scholar Theses

Somatic mutations may drive tumorigenesis or lead to new, immunogenic epitopes (neoantigens). The immune system is thought to represses neoplastic growths through the recognition of neoantigens presented only by tumor cells. To study mutations as well as the immune response to mutation-generated antigens, we have created a conditional knockin mouse line with a gene encoding, 5’ to 3’, yellow fluorescent protein (YFP), ovalbumin (which is processed to the immunologically recognizable peptide, SIINFEKL), and cyan fluorescent protein (CFP), or, YFP-ovalbumin-CFP. A frame shift mutation has been created at the 5’ end of the ovalbumin gene, hence YFP should always be expressed, …

Redox Regulation Of Protein Translation In Eukaryotes, Maxim Gerashchenko

Department of Biochemistry: Dissertations, Theses, and Student Research

Gene expression may be controlled at multiple levels, e.g., through genomic architecture, transcription and translation. In the current work, we focused on regulation of protein synthesis. Historically, the investigation of the regulation of gene expression at the level of translation lagged behind the transcriptional control because of the lack of accessible high-throughput methods. Our research has begun with the finding of the use of alternative non-AUG start codon in thioredoxin-glutathione reductase (TGR), a selenoprotein involved in redox control during male reproduction. The use of this codon, CUG, relies on the Kozak consensus sequence and ribosomal scanning mechanism. However, the CUG …

Lineage-Specific Transcriptional Profiles Of Symbiodinium Spp. Unaltered By Heat Stress In A Coral Host, Daniel J. Barshis, Jason T. Ladner, Thomas A. Oliver, Stephen R. Palumbi

Biological Sciences Faculty Publications

Dinoflagellates of the genus Symbiodinium form an endosymbiosis with reef building corals, in which photosynthetically derived nutrients comprise the majority of the coral energy budget. An extraordinary amount of functional and genetic diversity is contained within the coral-associated Symbiodinium, with some phylotypes (i.e., genotypic groupings), conferring enhanced stress tolerance to host corals. Recent advances in DNA sequencing technologies have enabled transcriptome-wide profiling of the stress response of the cnidarian coral host; however, a comprehensive understanding of the molecular response to stress of coral-associated Symbiodinium, as well as differences among physiologically susceptible and tolerant types, remains largely unexplored. Here, …

Identification Of Set1 Target Genes, William Beyer, Scott D. Briggs

The Summer Undergraduate Research Fellowship (SURF) Symposium

The Set1 complex, a histone methyltransferase complex found in S. cerevisiae (budding yeast), is the only histone methyltransferase responsible for catalyzing methylation of histone H3 at Lysine 4. It possesses homologues in other species, humans included. While yeast only have the Set1 complex, the human homologues of the yeast Set1 complex include mixed-lineage leukemia family (MLL1-4), Set1 A, Set1 B, among others. MLL1-4 has been shown to play a role in transcription, cell type specification, and the development of leukemia. One application of characterizing the role of a protein is that the information gained can provide insight into the function …

Saccharomyces Cerevisiae Cdc7 Homology In Drosophila Melanogaster, Marcus R. Hosler, Robert E. Stephenson, Vikki M. Weake

The Summer Undergraduate Research Fellowship (SURF) Symposium

Saccharomyces cerevisiae Dbf4(Dumbbell former 4) and Cdc7(Cell Division Cycle 7) form a complex that phosphorylates Mcm2 (Minichromosome maintenance 2) to initiate DNA replication. Cdc7 is a target for cancer research because there is a Cdc7 ortholog in humans that is necessary for DNA replication and cell survival. Our goal is to characterise a putative Cdc7 homolog in Drosophila melanogaster (dCdc7). We have previously shown that expression of the known Drosophila Dbf4 ortholog, Chiffon, and dCdc7 can rescue yeast cells deficient in active Cdc7. Our hypothesis is that the dCdc7 is activated by Chiffon to phosphorylate MCM2. To test this hypothesis, …

Effects Of Ergot Alkaloids And Bovine Bodily Fluids On Cytochrome P450 Activity, Nicholas Scott Ezell

Graduate Theses and Dissertations

This thesis evaluates the Promega^TM P450-Glo assay (Promega^TM V9800) as a tool for quantifying ergot alkaloid concentration. Current techniques used for detection of ergot alkaloids are slow and expensive, do not detect all ergot alkaloids, or are not effective on bovine bodily fluids. The first study was conducted to determine effects of commercial ergot alkaloids (n = 6; 0 - 400 μM) on the Promega^TM P450-Glo assay. Cytochrome P450 (CYP450) activity in assay had a differential response to each ergot alkaloid and concentration. As concentrations of ergotamine, dihydroergotamine, ergocornine, and ergocryptine increased CYP450 activity was inhibited (P …

The Role Of Cardiolipin In Iron Homeostasis And Glutathione Metabolism, Vinay A. Patil

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. Inside the mitochondria, CL is a critical target of mitochondrial generated reactive oxygen species (ROS) and regulates signaling events related to apoptosis and aging. CL deficiency causes perturbation of signaling pathways outside the mitochondria, including the PKC-Slt2 cell integrity pathway and the high osmolarity glycerol (HOG) pathway, and is a key player in the cross-talk between the mitochondria and the vacuole. The importance of CL in human health is underscored by the observation that perturbation of CL …

Aβ Alters The Dna Methylation Status Of Cell-Fate Genes In An Alzheimer’S Disease Model, Gary D. Isaacs, Noor Taher, Courtney Mckenzie, Rebecca Garrett, Matthew Baker, Nena Fox

Faculty Publications and Presentations

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-β plaques (Aβ). Despite ongoing research, some ambiguity remains surrounding the role of Aβ in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aβ treatment. We isolated the DNA from neurons treated with Aβ or vehicle, and digested the two samples with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. …

Investigation Of Bacterial Rna-Directed Dna Methylation Via Dcm And Hfq, Dandan Li

Wayne State University Theses

Bacterial small RNAs and the RNA chaperone Hfq play crucial roles in post-transcriptional gene regulation, often as parts of stress-response pathways, but little is known about their roles in regulation of gene transcription. A recent report showed that changes in methylation patterns caused by DNA cytosine methyltransferase (Dcm) were linked to gene regulation occurring during the transition to stationary phase. Here, we show that Dcm involves in the stress responses under nutrient starvation and cold stress. Dcm and Hfq together mediate gene expression under cold stress. Hfq promotes Dcm-catalyzed cytosine methylation at specific sites near the rpoS promoter, which is …

Electroporation-Mediated Gene Transfer Directly To The Swine Heart, Barbara Hargrave, Harre Downey, Cathryn Lundberg, Annelise Israel, Yeong-Jer Chen, Richard Heller

Bioelectrics Publications

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on …

Malnutrition In Sickle Cell Anemia: Implications For Infection, Growth And Maturation, Hyacinth I. Hyacinth, Oluwatoyosi A. Adekeye, Christopher S. Yilgwan

Journal of Social, Behavioral, and Health Sciences

Sickle cell anemia (SCA) is a genetic disease that affects mostly individuals of African and/or Hispanic descent, with the majority of cases in sub-Saharan Africa. Individuals with this disease show slowed growth, delayed sexual maturity, and poor immunologic function. These complications could partly be explained by the state of undernutrition associated with the disease. Proposed mechanism of undernutrition include protein hypermetabolism, decreased dietary intake possibly from interleukin-6-related appetite suppression, increased cardiac energy demand/expenditure, and increased red cell turnover. All the above mechanisms manifest as increased resting energy expenditure. Nutritional intervention utilizing single or multiple nutrient supplementation has led to improved …

A Genetic, Transgenic, And Transcriptomic Analysis Of Larval Salivary Gland Physiology In Drosophila Melanogaster, Elana A. Paladino

UNLV Theses, Dissertations, Professional Papers, and Capstones

Cholesterol is the precursor to a unique class of lipophilic signaling molecules called steroid hormones that initiate the development of sexual characteristics, reactions to stress, and maintenance of metabolism, among many other functions. Although much progress has been made in understanding the function of these signaling hormones, we do not fully understand how a single steroid can cause many distinct, tissue-specific responses. Drosophila melanogaster is an effective model for understanding steroid hormone action because of its simplicity. The steroid molting hormone 20-hydroxyecdysone (hereafter, 20E) is the primary active steroid in Drosophila and mediates not only larval molts and the emergence …

Mutation And Complementation Of A Cellulose Synthase (Cesa) Gene, Ahmed Y. El-Araby

Senior Honors Projects

Cellulose is a carbohydrate polymer that is composed of repeating glucose subunits. Being the most abundant organic compound in the biosphere and comprising a large percentage of all plant biomass, cellulose is extremely plentiful and has a significant role in nature. Cellulose is present in plant cell walls, in commercial products such as those made from wood or cotton, and is of interest to the biofuel industry as a potential alternative fuel source. Although indigestible by humans, cellulose is nutritionally valuable, serving as a dietary fiber. Because of its ubiquity and importance in many areas, studying cellulose will prove to …

Heterotopic Ossification: Cellular Basis, Symptoms, And Treatment, Brian Wolfe

Senior Honors Theses

Heterotopic ossification (HO) is the process by which calcified bone develops in soft tissues. Because of the abnormal calcification, complications such as bone deformation, loss of range of motion, and joint immobility adversely affect patients. There are many genetic types of heterotopic ossification, namely fibrodysplasia ossificans progressiva, progressive osseous heteroplasia, and Albright hereditary osteodystrophy. However, this condition can also arise from surgery, burns, or traumatic injuries, so it is seen as an important area for research in the future. There are various treatments available such as non-steroidal anti-inflammatory drugs and radiation therapy, as well as combinations of the two. The …

Genetic Control Of A Central Pattern Generator: Rhythmic Oromotor Movement In Mice Is Controlled By A Major Locus Near Atp1a2, Steven J. St. John, John D. Boughter Jr, Megan K. Mulligan, Kenichi Tokita, Lu Lu, Detlef H. Heck, Robert W. Williams

Faculty Publications

calreticulin, Animals, Chromosome Mapping, Mammalian Chromosomes, Gene Expression Regulation, Genetic Linkage, Genome-Wide Association Study. Inbred C57BL Mice, Inbred DBA Mice, Quantitative Trait Loci, Sodium-Potassium-Exchanging ATPase/genetics, Atp1a2 protein, Sodium-Potassium-Exchanging ATPase, feeding behavior, drinking behavior, mice, central pattern generator, genetic control

Expansion Dating: Calibrating Molecular Clocks In Marine Species From Expansions Onto The Sunda Shelf Following The Last Glacial Maximum, Eric D. Crandall, Elizabeth J. Sbrocco, Timery S. Deboer, Paul H. Barber, Kent E. Carpenter

Biological Sciences Faculty Publications

The rate of change in DNA is an important parameter for understanding molecular evolution and hence for inferences drawn from studies of phylogeography and phylogenetics. Most rate calibrations for mitochondrial coding regions in marine species have been made from divergence dating for fossils and vicariant events older than 1-2 My and are typically 0.5-2% per lineage per million years. Recently, calibrations made with ancient DNA (aDNA) from younger dates have yielded faster rates, suggesting that estimates of the molecular rate of change depend on the time of calibration, decaying from the instantaneous mutation rate to the phylogenetic substitution rate. aDNA …

Dna Secondary Structures And Their Contribution To Mutagenesis In B. Subtilis Stationary Phase Cells, Carmen Vallin, Holly Martin, Christian Ross, Ronald Yasbin, Eduardo Robleto

Undergraduate Research Opportunities Program (UROP)

It is widely known and accepted that the cause of many mutations in cells are generated during the replication process of actively dividing cells, however more recent research has shown that mutations also arise in non growing conditions, a phenomenon known as stationary phase mutagenesis. Much of what is known come from studies in eukaryotic and bacterial models. It has been proposed that in non~growing cells, the process of transcription plays an important role in mutagenesis. We test the hypothesis that DNA secondary structures, formed during transcription, promote mutagenesis. The transcription-generated structures are speculated to be prone to mutations by …

Evaluation Of Delivery Conditions For Cutaneous Plasmid Electrotransfer Using A Multielectrode Array, Bernadette Ferraro, Loree C. Heller, Yolmari L. Cruz, Siqi Guo, Amy Donate, Richard Heller

Bioelectrics Publications

Electroporation (EP) is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues. Delivery of plasmid DNA by EP to a large surface area is not practical because the distance between the electrode pairs, and therefore the applied voltage, must be increased to effectively permeabilize the cell membrane. The design of the multielectrode array (MEA) incorporates multiple electrode pairs at a fixed distance to allow for delivery of plasmid DNA to the skin, potentially reducing the sensation associated with in vivo EP. In this report, we evaluate the effects of field …

Loss Of Bloom Syndrome Protein Causes Destabilization Of Genomic Architecture And Is Complemented By Ectopic Expression Of Escherichia Coli Recg In Human Cells, Michael Wayne Killen

University of Kentucky Doctoral Dissertations

Genomic instability driven by non-allelic homologous recombination (NAHR) provides a realistic mechanism that could account for the numerous chromosomal abnormalities that are hallmarks of cancer. We recently demonstrated that this type of instability could be assayed by analyzing the copy number variation of the human ribosomal RNA gene clusters (rDNA). Further, we found that gene cluster instability (GCI) was present in greater than 50% of the human cancer samples that were tested. Here, data is presented that confirms this phenomenon in the human GAGE gene cluster of those cancer patients. This adds credence to the hypothesis that NAHR could be …

Elucidating Functional Roles For Myogenin In Adult Skeletal Muscle Metabolism, Exercise Capacity, And Regeneration, Jesse Flynn

Dissertations and Theses (Open Access)

The four basic helix-loop-helix myogenic transcription factors, myogenin, Myf5, MRF4, and MyoD are critical for embryonic skeletal muscle development. Myogenin is necessary for the terminal differentiation of myoblasts into myofibers during embryogenesis, but little is known about the roles played by myogenin in adult skeletal muscle function and metabolism. Furthermore, while metabolism is a well-studied physiological process, how it is regulated at the transcriptional level remains poorly understood. In this study, my aim was to determine the function of myogenin in adult skeletal muscle metabolism, exercise capacity, and regeneration. To investigate this, I utilized a mouse strain harboring the Myogflox …

Physical Interaction Between Vivid And White Collar Complex Regulates Photoadaptation In Neurospora, Chen-Hui H. Chen, Bradley S. Demay, Amy S. Gladfelter, Jay Dunlap, Jennifer J. Loros

Dartmouth Scholarship

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa, VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora. Using two previously characterized VVD mutants, we show …