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

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

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 terms1

Key Terms

  • Bipolar Spindle Assembly

Mammalian spindle


Key terms2

Key Terms

  • Oligonucleotide

  • Plasmid

    • Insert

    • Vector

  • MTOC (Microtubule Organizing Center)


Review of literature

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 literature1

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

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.


Kinesin 14 tail domain dd 35da4b

DD/


Materials and methods

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 methods1

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

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 methods2

Materials and Methods

  • Quick Screen DNA Analysis

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

Leilani Cruz Final Report SURP


Materials and methods3

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 methods4

Materials and Methods

  • Yeast Transformation

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

Leilani Cruz Final Report SURP


Materials and methods5

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

Predicted Results

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

DD/


Conclusion

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

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

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

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

Questions?


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