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Modeling of the 5 Nontranslated Region of Coxsackievirus B1

Introduction. CoxsackievirusRNA Secondary StructuresComputer ModelingAnalyzing ResultsEnzymatic Probing. Enterovirus. Small virus made of protein and RNAPoliovirus, coxsackievirus, echovirus3 Forms of Poliovirus23 Coxsackie A viruses6 Coxsackie B viruses28 Echoviruses4 other Enteroviruses

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Modeling of the 5 Nontranslated Region of Coxsackievirus B1

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    1. Modeling of the 5 Nontranslated Region of Coxsackievirus B1 Wade Schulz Biology Colloquium Spring 2003

    2. Introduction Coxsackievirus RNA Secondary Structures Computer Modeling Analyzing Results Enzymatic Probing

    3. Enterovirus Small virus made of protein and RNA Poliovirus, coxsackievirus, echovirus 3 Forms of Poliovirus 23 Coxsackie A viruses 6 Coxsackie B viruses 28 Echoviruses 4 other Enteroviruses Second most common viral infection Led by rhinovirus (common cold)

    4. Associated Infections Aseptic meningitis, encephalitis Myocarditis, Dilated Cardiomyopathy Type I diabetes Amyotrophic Lateral Sclerosis (ALS) Post-Polio Syndrome Chronic Fatigue Syndrome

    5. Coxsackievirus B1 Two types used in experiment 1.24 Wild type virus Causes acute infection as well as chronic disease 2.17 Mutated form of virus Causes acute infection but no chronic disease

    6. Mouse Model

    7. Mutational Change What changes does the mutation cause in the viral structure Does the structural change affect regulation

    8. RNA Secondary Structures Caused by bonding between bases on RNA Creates stems and loops in RNA Double-stranded stem Single-stranded loop

    9. Computer Modeling MFold Created by Michael Zuker Uses algorithms to compute lowest energy models to create structures Also known as a squiggles plot GeneQuest Part of Lasergene Suite Only provides folding for one energy level

    10. Computer Modeling RNA sequence is entered into database Folding conditions are set 37C, energy increments Server folds based on Zuker or other algorithms

    11. Data Analysis Stems and loops were identified Beginning and ending nucleotides were recorded Most common stems are assumed to exist Enzymatic probing done to determine actual structure

    12. Data Analysis

    13. Data Analysis

    14. Stem Changes Repeated stem changes were noticed between 1.24 and 2.17 structures Change was noted in stem where mutation was present

    15. Enzymatic Probing: Primer Extension Use enzymes to cleave RNA at specific points (single strands) RNase T1 - Guanosine RNase U2 - Adenosine RNase A - Pyrimidines RNA then placed in gel to determine lengths

    16. Primer Extension and Gel Analysis

    17. Conclusions 1. Presumptive evidence from computer modeling that 1.24 and 2.17 forms differ 2. Stem and loop just upstream of translational start may function in regulation and tropism

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