Genetic Structures Commons^™

A "Choose-Your-Own" Classroom-Based Activity That Promotes Scientific Inquiry About Rna Interference, Jeremy L. Hsu

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

RNA interference (RNAi), the process that results in the degradation of a target gene’s mRNA, is a fundamental part of eukaryotic gene regulation and is also an important molecular technique that allows for experimental manipulation of gene expression without altering DNA sequences. Despite the importance of RNAi, there have been relatively few lecture-based activities designed to teach about the consequences of this process and counter common misconceptions. I present here an inquiry-based activity that is centered around a “choose your own experiment” design where students generate hypotheses and critically evaluate their ideas by choosing several simulated experiments. The activity presents …

Fine Mapping And Identification Of Serum Urate Loci In American Indians: The Strong Heart Family Study, Geetha Chittoor, Karin Haack, Poojitha Balakrishnan, Christopher Bizon, Sandra Laston, Lyle G. Best, Jean W. Maccluer, Kari E. North, Jason G. Umans

School of Medicine Publications

While studies have reported genetic loci affecting serum urate (SU) concentrations, few studies have been conducted in minority populations. Our objective for this study was to identify genetic loci regulating SU in a multigenerational family-based cohort of American Indians, the Strong Heart Family Study (SHFS). We genotyped 162,718 single nucleotide polymorphisms (SNPs) in 2000 SHFS participants using an Illumina MetaboChip array. A genome-wide association analysis of SU was conducted using measured genotype analysis approach accounting for kinships in SOLAR, and meta-analysis in METAL. Our results showed strong association of SU with rs4481233, rs9998811, rs7696092 and rs13145758 (minor allele frequency (MAF) …

Integration Of Random Forest Classifiers And Deep Convolutional Neural Networks For Classification And Biomolecular Modeling Of Cancer Driver Mutations, Steve Agajanian, Odeyemi Oluyemi, Gennady M. Verkhivker

Mathematics, Physics, and Computer Science Faculty Articles and Research

Development of machine learning solutions for prediction of functional and clinical significance of cancer driver genes and mutations are paramount in modern biomedical research and have gained a significant momentum in a recent decade. In this work, we integrate different machine learning approaches, including tree based methods, random forest and gradient boosted tree (GBT) classifiers along with deep convolutional neural networks (CNN) for prediction of cancer driver mutations in the genomic datasets. The feasibility of CNN in using raw nucleotide sequences for classification of cancer driver mutations was initially explored by employing label encoding, one hot encoding, and embedding to …

Heterogeneity Of Disease-Causing Variants In The Swedish Galactosemia Population: Identification Of 16 Novel Galt Variants, Annika Ohlsson, Mary Hunt, Anna Wedell, Ulrika Von Döbeln

Articles

The aim was to determine disease-causing variants in the GALT gene which codes for the enzyme galactose-1-phosphate uridylyltransferase. Loss of activity of this enzyme causes classical galactosemia-a life threatening, treatable disorder, included in the Swedish newborn screening program since 1967. A total of 66 patients with the disease are known in Sweden and 56 index patients were investigated. An additional two patients with Duarte galactosemia were included. The disease-causing variants were identified in all patients. As reported from other countries only a few variants frequently recur in severe disease. The two variants p.(Gln188Arg) (c.563A>G) and p.(Met142Lys) (c.425T>A) are …

Kinetics Of Dextromethorphan-O-Demethylase Activity And Distribution Of Cyp2d In Four Commonly-Used Subcellular Fractions Of Rat Brain, Barent N. Dubois, Farideh Amirrad, Reza Mehvar

Pharmacy Faculty Articles and Research

The purpose of this study was to compare the enzymatic kinetics and distribution of cytochrome P450 2D (CYP2D) among different rat brain subcellular fractions.

Rat brains were used to prepare total membrane, crude mitochondrial, purified mitochondrial, and microsomal fractions, in addition to total homogenate. Michaelis–Menten kinetics of the brain CYP2D activity was estimated based on the conversion of dextromethorphan (DXM) to dextrorphan using UPLC-MS/MS. Protein levels of CYP2D and subcellular markers were determined by Western blot.

Microsomal CYP2D exhibited high affinity and low capacity, compared with the mitochondrial CYP2D that had a much lower (∼50-fold) affinity but a higher (∼six-fold) …

Optimisation Of Ion Exchange Chromatography Purification Protocols For A Staphylococcal Peptidoglycan Degrading Hydrolase Enzyme, Fiona Maher

Theses

Bacteriophage (phage) are the most abundant biological entities on earth and were first discovered by d’Herelle in 1917. They are found wherever their hosts live and, like all viruses they do not have the ability to make their own protein. Therefore, in order to reproduce, phage must invade and infect bacterial cells. This project focused on the optimisation of Ion Exchange Chromatography purification protocols for a staphylococcal peptidoglycan degrading hydrolase enzyme (CHAPk). The project objective was to obtain the greatest yield of enzyme from the growth of the E.coU XL 1-Blue expression system into which the vector pQE60 was previously …

Activity Of Distinct Growth Factor Receptor Network Components In Breast Tumors Uncovers Two Biologically Relevant Subtypes, Moom Roosan, Shelley M. Macneil, David F. Jenkins, Gajendra Shrestha, Sydney R. Wyatt, Jasmine A. Mcquerry, Stephen R. Piccolo, Laura M. Heiser, Joe W. Gray, W. Evan Johnson, Andrea H. Bild

Pharmacy Faculty Articles and Research

Background
The growth factor receptor network (GFRN) plays a significant role in driving key oncogenic processes. However, assessment of global GFRN activity is challenging due to complex crosstalk among GFRN components, or pathways, and the inability to study complex signaling networks in patient tumors. Here, pathway-specific genomic signatures were used to interrogate GFRN activity in breast tumors and the consequent phenotypic impact of GRFN activity patterns.

Methods
Novel pathway signatures were generated in human primary mammary epithelial cells by overexpressing key genes from GFRN pathways (HER2, IGF1R, AKT1, EGFR, KRAS (G12V), RAF1, BAD). The pathway analysis toolkit Adaptive Signature Selection …

P33. Design And Evaluation Of An Escherichia Coli Biomarker For Indication Of Ph, Kevin X. Zhou, Luana Langlois, Ashmita Singh, John Prince

Western Research Forum

Measuring pH is one of the most commonly used techniques in both the laboratory as well as the field due to its importance in a multitude of biochemical processes. Traditional methods of measuring pH may be highly developed in accuracy and precision but often involve disruption of the environment. Biological markers offer an alternative that allows for long-term pH monitoring. This innovative approach allows for vast applications such as in the manufacturing, food processing and research industries. Under moderate acidic conditions, the asr (acid shock RNA) gene is highly inducible and has been demonstrated to be crucial for growth at …

Multipoint Genome-Wide Linkage Scan For Nonword Repetition In A Multigenerational Family Further Supports Chromosome 13q As A Locus For Verbal Trait Disorders, D. T. Truong, L. D. Shriberg, S. D. Smith, K. L. Chapman, A. R. Scheer-Cohen, M. M.C. Demille, A. K. Adams, Alejandro Q. Nato Jr., E. M. Wijsman, J. D. Eicher, J. R. Gruen

Biochemistry and Microbiology

Verbal trait disorders encompass a wide range of conditions and are marked by deficits in five domains that impair a person’s ability to communicate: speech, language, reading, spelling, and writing. Nonword repetition is a robust endophenotype for verbal trait disorders that is sensitive to cognitive processes critical to verbal development, including auditory processing, phonological working memory, and motor planning and programming. In the present study, we present a six-generation extended pedigree with a history of verbal trait disorders. Using genome-wide multipoint variance component linkage analysis of nonword repetition, we identified a region spanning chromosome 13q14–q21 with LOD = 4.45 between …

Lack Of Association Between Slc30a8 Variants And Type 2 Diabetes In Mexican American Families, Hemant Kulkarni, Manju Mamtani, Juan M. Peralta, Vincent P. Diego, Thomas D. Dyer, Harald H. H. Goring, Laura Almasy, Sarah Williams-Blangero, Michael C. Mahaney, Ravindranath Duggirala, Joanne E. Curran, John Blangero

School of Medicine Publications

SLC30A8 encodes zinc transporter 8 which is involved in packaging and release of insulin. Evidence for the association of SLC30A8 variants with type 2 diabetes (T2D) is inconclusive. We interrogated single nucleotide polymorphisms (SNPs) around SLC30A8 for association with T2D in high-risk, pedigreed individuals from extended Mexican American families. This study of 118 SNPs within 50 kb of the SLC30A8 locus tested the association with eight T2D-related traits at four levels: (i) each SNP using measured genotype approach (MGA); (ii) interaction of SNPs with age and sex; (iii) combinations of SNPs using Bayesian Quantitative Trait Nucleotide (BQTN) analyses; and (iv) …

Genetic Candidate Variants In Two Multigenerational Families With Childhood Apraxia Of Speech, Peter Beate, Ellen M. Wijsman, Alejandro Q. Nato Jr., University Of Washington Center For Mendelian Genomics, Mark M. Matsushita, Kathy L. Chapman, Ian B. Stanaway, John Wolff, Kaori Oda, Virginia B. Gabo, Wendy H. Raskind

Biochemistry and Microbiology

Childhood apraxia of speech (CAS) is a severe and socially debilitating form of speech sound disorder with suspected genetic involvement, but the genetic etiology is not yet well understood. Very few known or putative causal genes have been identified to date, e.g., FOXP2 and BCL11A. Building a knowledge base of the genetic etiology of CAS will make it possible to identify infants at genetic risk and motivate the development of effective very early intervention programs. We investigated the genetic etiology of CAS in two large multigenerational families with familial CAS. Complementary genomic methods included Markov chain Monte Carlo linkage …

Identification Of Potential Drug Targets In Cancer Signaling Pathways Using Stochastic Logical Models, Peican Zhu, Hamidreza Montazeri Aliabadi, Hasan Uludag, Jie Han

Pharmacy Faculty Articles and Research

The investigation of vulnerable components in a signaling pathway can contribute to development of drug therapy addressing aberrations in that pathway. Here, an original signaling pathway is derived from the published literature on breast cancer models. New stochastic logical models are then developed to analyze the vulnerability of the components in multiple signalling sub-pathways involved in this signaling cascade. The computational results are consistent with the experimental results, where the selected proteins were silenced using specific siRNAs and the viability of the cells were analyzed 72 hours after silencing. The genes elF4E and NFkB are found to have nearly no …

Identity-By-Descent Estimation With Population- And Pedigree-Based Imputation In Admixed Family Data, Mohamad Saad, Alejandro Q. Nato Jr., Fiona L. Grimson, Steven M. Lewis, Lisa A. Brown, Elizabeth M. Blue, Timothy A. Thornton, Elizabeth A. Thompson, Ellen M. Wijsman

Biochemistry and Microbiology

Background: In the past few years, imputation approaches have been mainly used in population-based designs of genome-wide association studies, although both family- and population-based imputation methods have been proposed. With the recent surge of family-based designs, family-based imputation has become more important. Imputation methods for both designs are based on identity-by-descent (IBD) information. Apart from imputation, the use of IBD information is also common for several types of genetic analysis, including pedigree-based linkage analysis.

Methods: We compared the performance of several family- and population-based imputation methods in large pedigrees provided by Genetic Analysis Workshop 19 (GAW19). We also evaluated the …

Estimating Relationships Between Phenotypes And Subjects Drawn From Admixed Families., Elizabeth M. Blue, Lisa A. Brown, Matthew P. Conomos, Jennifer L. Kirk, Alejandro Q. Nato Jr., Alice B. Popejoy, Jesse Raffa, John Ranola, Ellen M. Wijsman, Timothy Thornton

Biochemistry and Microbiology

Background: Estimating relationships among subjects in a sample, within family structures or caused by population substructure, is complicated in admixed populations. Inaccurate allele frequencies can bias both kinship estimates and tests for association between subjects and a phenotype. We analyzed the simulated and real family data from Genetic Analysis Workshop 19, and were aware of the simulation model.

Results: We found that kinship estimation is more accurate when marker data include common variants whose frequencies are less variable across populations. Estimates of heritability and association vary with age for longitudinally measured traits. Accounting for local ancestry identified different true associations …

Molecular Genetics Of Ms4a6a And Alzheimer's Disease, Ryan Harpole

Lewis Honors College Capstone Collection

Increased Alzheimer’s disease (AD) risk has previously been associated with a SNP called rs610932 near the gene MS4A6A. The goal of this experiment was to quantify the expression of two MS4A6A isoforms in the brains of AD and non-AD subjects, particularly as a function of rs610932 genotype. According to an article titled “Alzheimer’s Disease Susceptibility Variants in the MS4A6A Gene are Associated with Altered Levels of MS4A6A Expression in Blood”, MS4A6A has four different isoforms that have been reported to be differentially expressed in the blood of AD subjects compared to non-AD subjects (Petroula et al., 2014). After statistically …

Deletion Of Shank1 Has Minimal Effects On The Molecular Composition And Function Of Glutamatergic Afferent Postsynapses In The Mouse Inner Ear, Jeremy P. Braude, Sarath Vijayakumar, Katherine Baumgarner, Rebecca Laurine, Timothy A. Jones, Sherri M. Jones, Sonya J. Pyott

Department of Special Education and Communication Disorders: Faculty Publications

Abstract

Shank proteins (1-3) are considered the master organizers of glutamatergic postsynaptic densities in the central nervous system, and the genetic deletion of either Shank1, 2, or 3 results in altered composition, form, and strength of glutamatergic postsynapses. To investigate the contribution of Shank proteins to glutamatergic afferent synapses of the inner ear and especially cochlea, we used immunofluorescence and quantitative real time PCR to determine the expression of Shank1, 2, and 3 in the cochlea. Because we found evidence for expression of Shank1 but not 2 and 3, we investigated the morphology, composition, and function of afferent postsynaptic densities …

Cryptorchidism And Infertility In Rats With Targeted Disruption Of The Adamts16 Locus, Shakila Abdul-Majeed, Blair Mell, Surya M. Nauli, Bina Joe

Pharmacy Faculty Articles and Research

A Disintegrin And Metalloproteinase with ThromboSpondin motifs16 (ADAMTS-16) is a member of a family of metalloproteinases. Using a novel zinc-finger nuclease based gene-edited rat model harboring a targeted mutation of the Adamts16 locus, we previously reported this gene to be linked to blood pressure regulation. Here we document our observation with this model that Adamts16 is essential for normal development of the testis. Absence of Adamts16 in the homozygous Adamts16(mutant) males resulted in cryptorchidism and male sterility. Heterozygous Adamts16(mutant) males were normal, indicating that this is a recessive trait. Testes of homozygous Adamts16(mutant) males were significantly smaller with significant histological …

Retinoic Acid Regulation Of Thyroid Hormone Action In Bone Cells, Anjali Babbar

Loma Linda University Electronic Theses, Dissertations & Projects

Retinoic acid and thyroid hormone are known to play key roles in the regulation of endochondral ossification. However, the issue of whether these two hormones interact with each other to regulate bone functions remains to be established. We investigated how thyroid hormone and retinoic acid interact to regulate cells involved in endochondral bone formation. We demonstrate that thyroid hormone treatment stimulates differentiation of ATDC5 chondrocytes and promotes formation of mineralized nodules while retinoic acid treatment at high dose inhibits chondrocyte differentiation and formation of mineralized nodules. Furthermore, thyroid hormone induced mineralized nodule formation is inhibited by co-treatment with retinoic acid …

Mapping Genes With Longitudinal Phenotypes Via Bayesian Posterior Probabilities, Anthony Musolf, Alejandro Q. Nato Jr., Douglas Londono, Lisheng Zhou, Tara C. Matise, Derek Gordon

Biochemistry and Microbiology

Most association studies focus on disease risk, with less attention paid to disease progression or severity. These phenotypes require longitudinal data. This paper presents a new method for analyzing longitudinal data to map genes in both population-based and family-based studies. Using simulated systolic blood pressure measurements obtained from Genetic Analysis Workshop 18, we cluster the phenotype data into trajectory subgroups. We then use the Bayesian posterior probability of being in the high subgroup as a quantitative trait in an association analysis with genotype data. This method maintains high power (>80%) in locating genes known to affect the simulated phenotype …

Truncation Of Type Iv Pilin Induces Mucoidy In Pseudomonas Aeruginosa Strain Pao579, T. Ryan Withers, F. Heath Damron, Yeshi Yin, Hongwei D. Yu

Biochemistry and Microbiology

Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ²²). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP …

Pitx2 Is Overexpressed In Follicular Cell-Derived Thyroid Cancer And Promotes Thyroid Cancer Proliferation By Regulating Cell Cycle, Yue Huang

Theses, Dissertations and Capstones

Thyroid cancer is the most prevailing malignancy of the endocrine system. Its incidence is rapidly rising at the second fastest rate of all malignancies in the United States, making it a significant health problem. Although the majority of thyroid cancer is slowly-growing and well-differentiated, available treatment options are very limited, and most of them require complete removal of the thyroid gland and surrounding tissues. Patients who have undergone thyroid removal have to take life-long hormone replacement therapy, which is very inconvenient and costly. Therefore, there is an urgent need to develop new treatments for this disease. As a prerequisite for …

Altered Self-Assembly And Apatite Binding Of Amelogenin Induced By N-Terminal Proline Mutation, Li Zhu, Vuk Uskoković, Thuan Le, Pamela Denbesten, Yulei Huang, Stefan Habelitz, Wu Li

Pharmacy Faculty Articles and Research

Objective—A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralized amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals.

Design—Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analyzed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM.

Results—As …

Hydrolysis Of Amelogenin By Matrix Metalloprotease-20 Accelerates Mineralization In Vitro, Vuk Uskoković, Feroz Khan, Haichuan Liu, Halina Ewa Witkowska, Li Zhu, Wu Li, Stefan Habelitz

Pharmacy Faculty Articles and Research

In the following respects, tooth enamel is a unique tissue in the mammalian body: (a) it is the most mineralized and hardest tissue in it comprising up to 95 wt% of apatite; (b) its microstructure is dominated by parallel rods composed of bundles of 40 – 60 nm wide apatite crystals with aspect ratios reaching up to 1:10,000 and (c) not only does the protein matrix that gives rise to enamel guides the crystal growth, but it also conducts its own degradation and removal in parallel. Hence, when mimicking the process of amelogenesis in vitro, crystal growth has to …

Biomimetic Precipitation Of Uniaxially Grown Calcium Phosphate Crystals From Full-Length Human Amelogenin Sols, Vuk Uskoković, Wu Li, Stefan Habelitz

Pharmacy Faculty Articles and Research

Human dental enamel forms over a period of 2 – 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of a dense amelogenin matrix is presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aims to establish the physicochemical and biochemical conditions for the synthesis of fibrous apatite crystals under the control of a recombinant fulllength human amelogenin matrix in combination with a programmable titration system. The growth …

Aging And Environmental Exposures Alter Tissue-Specific Dna Methylation Dependent Upon Cpg Island Context, Brock C. Christensen, E Andres Houseman, Carmen J. Marsit, Shichun Zheng, Margaret R. Wrensch, Joseph L. Wiemels, Heather H. Nelson, Margaret R. Karagas

Dartmouth Scholarship

Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci …

Genetic Mapping Of Secretion And Functional Determinants Of The Vibrio Cholerae Tcpf Colonization Factor, Shelly J. Krebs, Thomas J. Kirn, Ronald K. Taylor

Dartmouth Scholarship

Colonization of the human small intestine by Vibrio cholerae requires the type IV toxin-coregulated pilus (TCP). TcpF, which is encoded within the tcp operon, is secreted from the bacterial cell by the TCP apparatus and is also essential for colonization. Bacteria lacking tcpF are deficient in colonization, and anti-TcpF antibodies are protective in the infant mouse cholera model. In order to elucidate the regions of the protein that are required for secretion through the TCP apparatus and for its function in colonization, random mutagenesis of tcpF was performed. Analysis of these mutants suggests that multiple regions throughout the protein influence …

Identification And Characterization Of Novel Sir3/Mecp2-Chromatin Interactions, Nicholas L. Adkins

Theses, Dissertations and Capstones

The eukaryotic genome is packaged into chromosomes that are made up of a highly organized and heavily regulated structure called chromatin. The proteins involved in the compaction of DNA into this condensed state are mostly understood at the level of the structure of the nucleosome. The higher order arrangement of chromatin and how it effects gene regulation is only partially understood and characterized. The compaction of nucleosomal arrays into 30-nm and higher structures are partially the responsibility of architectural, or structural, chromatin associated proteins. The following dissertation analyzes the individual chromatin contributions of two well studied architectural proteins, the yeast …

Detection Of Point Mutations In The Dystrophin Gene, John Pedretti

Theses : Honours

The dystrophin gene has been localised to Xp 21.1. Mutations of this gene can lead to the clinical manifestations of Duchenne and Becker muscular dystrophies (DMD/BMD). In the majority of DMD and BMD patients the disease-causing mutation is a deletion detectable by southern analysis or multiplex PCR, however in 30% of patients no deletion is observed using these conventional tests. Using PCR amplification of cDNA it was possible to detect a deletion in the product of the dystrophin gene of one such individual affected with BMD. It was then necessary to characterise the mutation in order to determine whether this …