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Kinesin-14 Tail Domain: DD/35DA4B

Kinesin-14 Tail Domain: DD/35DA4B. Dr. JL Paluh at College of Nanoscale Science and Engineering University at Albany SUNY Presented by: Laura Patrick. Key Terms. Kinesin-like Motor Protein Families Kinesin-5 Kinesin-14 SpPkl1 Schizosaccharomyces pombe (fission yeast)

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Kinesin-14 Tail Domain: DD/35DA4B

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  1. Kinesin-14 Tail Domain: DD/35DA4B Dr. JL Paluh at College of Nanoscale Science and Engineering University at Albany SUNY Presented by: Laura Patrick

  2. Key Terms • Kinesin-like Motor Protein Families • Kinesin-5 • Kinesin-14 • SpPkl1 Schizosaccharomyces pombe (fission yeast) • HsHSET Homo sapiens (Humans) Leilani Cruz Final Report SURP

  3. Key Terms • Bipolar Spindle Assembly Mammalian spindle

  4. Key Terms • Oligonucleotide • Plasmid • Insert • Vector • MTOC (Microtubule Organizing Center)

  5. Review of Literature • Paluh, J.L. (2008) Kinesin-14 leaps to pole position in bipolar spindle assembly. Chinese Journal of Cancer, 27(9): 1-5. • Rodriguez, A.S., Killilea, A.N., Batac, J., Filopei, J., Simeonov, D.R., Lin, I., and J.L. Paluh (2008) Protein complexes at the microtubule organizing center regulate bipolar spindle assembly. Cell Cycle. 7(9): 1246-1253.

  6. Review of Literature • Simeonov, D.R., Kenny, K., Seo, L., Moyer, A., and J. L. Paluh. (2009) Distinct Kinesin-14 Mitotic Mechanisms in Spindle Bipolarity. Cell Cycle, 8 (21): 3571-583. • Cruz, L. (2010) Defining Minimal Elements in a Novel Kinesin-14 Tail Domain for Spindle Pole Localization and γ-TuSC Regulation. Summer Undergrad Research Fellowship (SURP) final report.

  7. Hypothesis • Kinesin-14 Pkl1 Tail domain, DD/35DA4B, contains elements for localization to spindle poles and functional interactions with MTOC proteins that are needed for it to regulate spindle bipolarity.

  8. DD/

  9. Materials and Methods • PCR (Polymerase Chain Reaction) • Oligonucleotides contain changes • PCR allows amplified copies of the altered Tail region DNA, DD/35DA4B to be generated for cloning Puc18 DD DD

  10. Materials and Methods • Ligation Reaction • To join the two linear pieces of DNA: the insert (DD/35DA4B) and the intermediate vector, Puc-18. • The intermediate vector was used first to provide us with the correct cloning sites for later ligations. Insert: DD/35DA4B Plasmid Vector: Puc-18

  11. Material and Methods • Bacterial Transformation • To separate products of the ligation reaction: ie. successful clones from non-successful clones Bacterial Cell Plasmid Insert Vector

  12. Materials and Methods • Quick Screen DNA Analysis • To identify the correct ligation product amongst transformed bacterial colonies. Leilani Cruz Final Report SURP

  13. Materials and Methods • Correct clones, (DD/35DA4B Puc-18) were then cloned into the yeast vector: Prep81. • Identical experiments were repeated with the new vector Prep81continued through the Quick Screen DNA isolation procedure.

  14. Materials and Methods • Yeast Transformation • To test the in vivo function of altered Kinesin-14 proteins. Leilani Cruz Final Report SURP

  15. Materials and Methods • Serial Dilution Assay • Compare strains by plating decreasing cell numbers of each onto triplicate plates and monitoring growth at different temperatures against wild type (positive) and nonfunctional (negative) gene controls.

  16. Predicted Results • In construct DD/35DA4B deletion of region D is minimal we predict that the construct will be functional. DD/

  17. Conclusion • Kinesin-14 Pkl1 Tail domain contains critical elements in order to regulate spindle assembly. • Construct DDP6 proved that part of region 6 and possibly D are critical and can not be deleted. • Construct 35DA4B proved that regions A, 4, and B are not critical and can be deleted.

  18. Future Studies • Finish testing remaining constructs for functionality: • 3P56P • 3P6D • 3P56DC • DD • Examine localization and protein interactions between modified Kinesin-14 Pkl1 and γ-TuSC MTOC proteins.

  19. References • Paluh, J.L. (2008) Kinesin-14 leaps to pole position in bipolar spindle assembly. Chinese Journal of Cancer, 27(9): 1-5. • Rodriguez, A.S., Killilea, A.N., Batac, J., Filopei, J., Simeonov, D.R., Lin, I., and J.L. Paluh (2008) Protein complexes at the microtubule organizing center regulate bipolar spindle assembly. Cell Cycle. 7(9): 1246-1253. • Simeonov, D.R., Kenny, K., Seo, L., Moyer, A., and J. L. Paluh. (2009) Distinct Kinesin-14 Mitotic Mechanisms in Spindle Bipolarity. Cell Cycle, 8 (21): 3571-583. • Cruz, L. (2010) Defining Minimal Elements in a Novel Kinesin-14 Tail Domain for Spindle Pole Localization and γ-TuSC Regulation. Summer Undergrad Research Fellowship (SURP) final report. • Kollman, J.M., Zelter, A., Muller, E.G.D., Fox, B., Rice, L.M. Davis, T.N., and D.A. Agard (2008) The structue of the g-tubulin small complex: Implications of its architecture and flexibility for microtubule nucleation. Molec. Biol. Cell 19: 207-215.

  20. Acknowledgments • Dr. Janet L. Paluh, CNSE at Univ. at Albany SUNY • Leilani Cruz and Lonnie Seo, Rensselaer Polytechnic Institute • Ms. Gleason and Ms. Strauss • My Family

  21. Questions?

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