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The Construction of Mutations in the 3  Cyclization Sequence of Dengue Virus Genome, for the Study of Translation

The Construction of Mutations in the 3  Cyclization Sequence of Dengue Virus Genome, for the Study of Translation. Tari Tan Dr. Theo Dreher Dr. Connie Bozarth HHMI, Summer 2005. The Global Impact of Dengue Virus. Dengue Fever & Dengue Hemorrhagic Fever. Endemic in more

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The Construction of Mutations in the 3  Cyclization Sequence of Dengue Virus Genome, for the Study of Translation

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  1. The Construction of Mutations in the 3 Cyclization Sequence of Dengue Virus Genome, for the Study of Translation Tari Tan Dr. Theo Dreher Dr. Connie Bozarth HHMI, Summer 2005

  2. The Global Impact of Dengue Virus Dengue Fever & Dengue Hemorrhagic Fever Endemic in more than 100 countries 50 million cases each year Areas infested with Aedes aegypti Areas with Aedes aegypti and dengue epidemic activity

  3. Dengue: Viral Properties • Flavivirus Genome Ranges from 10-11 kilobases 5' nucleotide cap +ssRNA • Serotype 2 (DEN-2)

  4. Translation • DEN-2: +ssRNA • Viral Proteins are responsible for replication, assembly, maturation, and exit from the cell

  5. Conserved Features Dumbbells 1 and 2 (DB1, DB2) Stemloops A and B (SLA, SLB) 5' Cyclization Sequence (cCS1) 3' Cyclization Sequence (CS1)

  6. Kunjin Virus, observed effect of cCS1 and CS1 mutations on replication 3' mutant - no replication 5' mutant - no replication Deletion of cCS1 - no replication 5'+3' mutant restoring complementarity - delayed start, but efficient replication DEN-2, effect of cCS1 and CS1 mutations on translation Replace cCS1 with modified CS1 - little impact (efficient translation) Replace CS1 with modified cCS1 - only 19% translation 5'+3' mutant restoring complementarity - could not rescue translation Previous Studies Dr. Alexander KhromykhWei Wei Chiu

  7. Five Mutations 1) IS TRANSLATION SEQUENCE-SPECIFIC FOR CS1? 2) Does the degree of complementarity between cCS1 and CS1 affect translational efficiency?

  8. Definitions The DEN-2 construct into which the mutated sequences are ligated The fragment (or, in general terms, the DEN-2 construct containing the fragment) containing the mutated CS1, which is ligated into the vector The resultant vector+insert construct containing the mutated CS1 Vector Insert Mutant/Variant

  9. Methodology • DCLD WT (insert); DCLD ∆DB1+2(vector)

  10. Methodology (Cont.) • PCR amplification of mutated sequences, using DCLD WT

  11. Methodology (Cont.) • Digest Final PCR product with Hind III and Kpn I; isolate 474 bp fragment • Insert mutants into DCLD ∆DB1+2 (which has already been digested with Hind III and Kpn I) via a ligation • Ultimately, the mutants will be used to assess translation by measuring luciferase luminescence

  12. Troubleshooting First PCR Purify Megaprimer Second PCR Hind III/Kpn I Digest Purify Fragment Ligation with DCLD ∆DB1+2 Grow/Prep Colonies Hind III/Kpn I Digest (Select) Send For Sequencing PCR - poor yield Digests - not cutting Liquid Cultures - not growing / no plasmid

  13. Annealing Temperature/ Mg2+ concentration • New Oligos 60o C 2 1.5 .75 mM mM mM 65o C, 1.5 mM MgCl2 2nd PCR, Khromykh (DCLD-SnaB1/Not1 template, WW12 + Khromykh megaprimer) 2nd PCR, A-Rich (DCLD-SnaB1/Not1 template, WW12 + A-Rich megaprimer) D K L S 137 bp 128 bp K Gel-pure Megaprimers, (3 ul/ 30) 479 bp Final PCR product, “K” mutant PCR Modification

  14. Diagnostic Digests • Performed by Dr. Bozarth BUFFERS Buffer R (HindIII buffer) Buffer KpnI HindIII ENZYMES KpnI

  15. NH2 H Ampicillin N O ß -lactam H NH CH3 S O CH3 N OH O O Bacterial Growth

  16. Liquid Cultures & Loss of Plasmid Plate Investigation • Hard-to-clone sequences? • Mutants toxic to cells?

  17. ∆DB 1+2 DCLD Phosphatased, phenol-extracted, EtOH precipitated, gel-pure vectors (digested with Hind III and Kpn I) Vector Reconstruction • Problem with the vector? • Make ∆DB1+2, as well as DCLD

  18. I. PCR - poor yield Annealing Temperature: 60o C MgCl2 Concentration: 1.5 mM Re-designed oligos to be shorter: better yield II. Digests - not cutting Dr. Bozarth’s Trials: bad KpnI or Buffer KpnI (bought new enzyme and buffer) III. Liquid cultures not growing / Loss of plasmid DCLD as control: only controls grew (2 x YT not the problem) Streaking: selective growing, but still problems in liquid cultures Re-make vector, both ∆DB 1+2 and DCLD: under investigation These issues remain unresolved. Summary

  19. Acknowledgements • HHMI - Dr. Kevin Ahern • URISC • Dr. Theo Dreher • Dr. Connie Bozarth • Everyone else in the Dreher lab for helping me out Thank you very much!

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