Tesis bryan aedes

Myotonic Dystrophy:

The Structure of CUG Repeats in Solution

By

Brian Truong

A Thesis

Presented to the Department of Chemistry
and the Honors College of the University of Oregon
in partial fulfillment of the requirements
for the degree of
Bachelor of Arts

March 2007
An Abstract of the Thesis of
Brian Truong for the degree ofBachelor of Arts
in the Department of Chemistry to be taken March 2007
Title: Myotonic Dystrophy: The Structure of CUG Repeats in Solution

Approved: ______________________________________
Andy Berglund, Ph.D.

Myotonic dystrophy (DM), a genetic and neuromuscular disorder, is the most common form of adult-onset muscular dystrophyresulting in symptoms such as proximal muscle weakness, myotonia, iridescent cataracts and cardiac arrhythmia. Myotonic dystrophy type 1 (DM1), the most prevalent type of DM, is caused by a (CTG)n expansion in the 3’ untranslated region of the dystrophin myotonin protein kinase gene. At the RNA level, the CUG repeats form a stem-loop that is thought to be the pathogenic element. Previous work inthe Berglund Lab determined the crystal structure of CUG repeats and found these repeats form a structure similar to standard double-stranded A-form nucleic acid. However, since crystal structures may be misleading, we verify A-form conformation using a solution-based assay. A-form double-stranded RNA (dsRNA) is known to be cleaved into small RNA molecules in cells; therefore, the cellularmachinery that recognizes and cleaves these dsRNA should degrade expanded CUG repeats. To test this hypothesis, we use an enzyme called Dicer that recognizes and cleaves A-form dsRNA. The cleavage of r(CUG)54 by Dicer confirms CUG repeats adopt a conformation similar to A-form in solution. The structure of CUG repeats may therefore lead to the design of potential drugs for DM.

ACKNOWLEDGEMENTSFirst, I would like to thank Andy Berglund and Jill Murray for their expert guidance and encouragement. Second, I would like to thank my family and friends for their support and motivation. Third, I would like to thank the Muscular Dystrophy Association (MDA) for allowing me to meet the people who inspired me to pursue this research endeavor.
Table of Contents

Chapter Page

I.Introduction 6

II. Overview of Methods 19

III. Materials and methods 21

Molecular cloning 21
Enzymatic digestion 21
Transcription 21
Annealing 22
Dicer activity assay 22
Visualization 22

IV. Results 23

V. Discussion 25

VI. Works Cited 28

List of Figures

Figure Page
1. CTG repeat counts on the DMPK gene 9
2. Watson-Crickbase-pairing 10
3. Nucleic acid structure 11
4. CUG repeat stem-loop structure 12
5. CUG repeats with U-U mismatches 13
6. Similarity of r(CUG)6 to A-form RNA 14
7. The central dogma of molecular biology 16
8. CUG repeat stem-loop structure with sequestered MBNL 17
9. Overview of experiment design 20
10. Annealing assay 23
11. Dicer activity assay 24

I. IntroductionMyotonic dystrophy (DM), the most common form of adult-onset muscular dystrophy, is a neuromuscular disease that affects approximately 1 in every 8,000 individuals and is characterized by a defective gene that is passed from one generation to the next1. DM is progressive, so the characteristic symptoms of muscle wasting and weakness develop gradually over the lifetime of an affected individual.In addition to general muscle weakness, people with DM also have myotonia, the delayed relaxation of skeletal muscles following voluntary contractions. While this is a mild problem in early onset patients, affected individuals eventually lose the ability to release the hand from a grip due to the myotonia2. Besides the muscles in the hands, the first muscles to be affected are usually those of…

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