Kinesin 14 tail domain dd 35da4b
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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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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)

      • HsHSET Homo sapiens (Humans)

Leilani Cruz Final Report SURP


Key Terms

  • Bipolar Spindle Assembly

Mammalian spindle


Key Terms

  • Oligonucleotide

  • Plasmid

    • Insert

    • Vector

  • MTOC (Microtubule Organizing Center)


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.


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.


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.


DD/


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


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


Material and Methods

  • Bacterial Transformation

    • To separate products of the ligation reaction:

      ie. successful clones from non-successful clones

Bacterial Cell

Plasmid

Insert

Vector


Materials and Methods

  • Quick Screen DNA Analysis

    • To identify the correct ligation product amongst transformed bacterial colonies.

Leilani Cruz Final Report SURP


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.


Materials and Methods

  • Yeast Transformation

    • To test the in vivo function of altered Kinesin-14 proteins.

Leilani Cruz Final Report SURP


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.


Predicted Results

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

DD/


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.


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.


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.


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


Questions?


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