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REU Final PowerPoint


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REU Final PowerPoint

  1. 1. An Investigation of Two Putative Peroxisomal Proteins: AtFHIT and AtHINT2 Alyssa Castle1, Ali Dorchak2, Laura Olsen2 1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan
  2. 2. Peroxisomes Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.  Small membrane bound organelles  Found in all eukaryotic cells  Responsible for many metabolic functions  Catabolism of Long Chain Fatty Acids  Metabolism of H2O2  Many peroxisomal disorders, usually resulting in death in early stages of life  Zellweger Syndrome  X-Linked Adrenoleuknodystrophy  D-Bifunctional Protein Deficiency
  3. 3. Peroxisomal Targeting Signals  Peroxisomes lack their own genome  Proteins are synthesized in the cytosol, then transported into the peroxisome  In order for proteins to be imported into peroxisomes, they must have one or both PTS1 (Carboxyl-terminal) or PTS2 (Amino-terminal) signals  Like a zip code
  4. 4. Question/Hypothesis  Do the AtFHIT and AtHINT2 encoding proteins localize to the peroxisome?  Hypothesize the proteins may localize in the peroxisome because other proteins within this ‘family’ have been shown to be peroxisomal.  HIT1 (PTS1), HIT2 (PTS2), HIT3 (PTS2) (Reumann S et al. Plant Physiol. 2009;150:125-143)
  5. 5. AtFHIT and AtHINT2  Putative PTS2 Signals
  6. 6. PTS2 Import Pathway  Once the protein is translocated into the peroxisomal matrix, the PTS2 signal at the amino terminus is cleaved by a protease (DEG15) upon import into the peroxisome.
  7. 7. Approach  Using 35S- radiolabeled protein
  8. 8. Methods  Lane 1 and 2 are the PCR amplified products of AtHINT2 and AtFHIT S P 6 T 7 Inserted Gene pCRII-TOPO  PCR products were then inserted into the pCRII-TOPO vector, the resulting plasmid was used to transform TOP10F’ E. coli
  9. 9. Methods Transformed cells were selected using blue-white screening Colonies were selected and the plasmid DNA was mini prepped Restriction digests of the mini prepped DNA were done to drop inserts out of the plasmid Lanes 4 and 5 show a successful drop of both genes out of pCRII- TOPO
  10. 10. Methods Clones showing a successful drop from the vector were sent for sequencing Maxi prepped clones and the plasmid DNA was purified by precipitation with PEG Linearization and purification Transcription Translation
  11. 11. Results AtFHIT AtHINT2 *Expected molecular mass= 20.4 kDa
  12. 12. Results 0 5 10 15 20 25 - + - + - + Corrected%Processed Substrate AtFHIT AtHINT2 AtASP3 Digital audioradiograph protease assay results
  13. 13. Conclusions  In vitro experiments with AtFHIT and AtHINT2 show minimal processing  Therefore are not very convincing whether they are peroxisomal  These results are not determinant of whether or not the proteins are peroxisomal  In vivo experiments would help to further analyze whether these proteins are peroxisomal
  14. 14. Acknowledgements Ali Dorchak Dr. Laura Olsen The Olsen Lab Dr. Aaron Wyman Dr. Cherie Dotson Dr. Ron Woodard University of Michigan College of Pharmacy The National Science Foundation
  15. 15. Questions?