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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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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