spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huang, H.-k.
Right arrow Articles by Hunter, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, H.-k.
Right arrow Articles by Hunter, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Isolation and characterization of the Pin1/Ess1p homologue in Schizosaccharomyces pombe

Han-kuei Huang1, Susan L. Forsburg1, Ulrik P. John2, Matthew J. O’Connell2,3 and Tony Hunter1,*

1 Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
2 Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett Street, Melbourne, VIC 8006, Australia
3 Department of Genetics University of Melbourne, Parkville, VIC 3052, Australia



View larger version (48K):

[in a new window]
  		Fig. 1. Protein sequence alignment of Pin1, dodo, Ess1p and Pin1p. The amino acid sequences are compared for maximal alignment. The shaded regions indicate identical amino acid residues among the four proteins.

 


View larger version (22K):

[in a new window]
  		Fig. 2. Cellular localization of Pin1p. (A) Schematic diagram of how the pin1+-GFP fusion chimera integrated into the chromosome. (B) The Pin1p-GFP expressing strain (Sp35) was grown to late log phase. Cells were fixed in 3.7% formaldehyde for 30 minutes followed by DAPI staining for 10 minutes in the dark. Cells were viewed with a Leitz Laborlux microscope and images were acquired by Adobe PhotoShop software using the SPOT-2 CCD digital camera.

 


View larger version (25K):

[in a new window]
  		Fig. 3. Overexpression of pin1+ causes a severe growth defect. (A) Wild-type (Sp13) and overexpression (Sp15) strains were grown on plates containing thiamine for two days and inoculated into non-repressive medium for 20 hours, which is the time required for induction of pin1+ overexpression. Cells with overexpressed pin1+ were re-inoculated into non-repressive medium to OD595=0.05 and cultured at 32°C. For every 12 hours, aliquots of cells were taken and the cell densities (OD595) were measured. (B) Same as A except that cell aliquots were fixed in 1 ml of cold 100% ethanol, resuspended in 50 mM sodium citrate and stained with 1 µM Sytox Green. Flow cytometry was performed with a Becton Dickinson FACScan and data were analyzed by CellQuest software.

 


View larger version (14K):

[in a new window]
  		Fig. 4. Deletion analysis of ESS1 and pin1+. (A) Wild-type diploid S. cerevisiae cells deleted for one copy of ESS1 were subjected to sporulation followed by tetrad dissection. Spores were grown on YEPD plates at 17°C for 9 days (left panel), or at 30°C for 4 days (middle panel), or at 37°C for 6 days (right panel). (B) A wild-type, two independent S. pombe pin1{Delta} strains and a rad3{Delta} strain were grown in YES medium overnight and then plated on YES plates followed by UV irradiation. Plates were cultured at 32°C with a foil cover for 1 day and without a foil cover for the subsequent 3 days. Colony numbers were calculated and survival rates were determined based on control plates, which were not irradiated. The data in this figure represent the average of three independent experiments with a standard deviation of less than 7%.

 


View larger version (87K):

[in a new window]
  		Fig. 5. Synthetic phenotypes of pin1 deletion and other mitotic mutants. Sp1(wild-type), Sp23 (pin1{Delta}), Sp62 (cdc25-22), Sp84(pin1{Delta} cdc25-22), Sp149 (pin1{Delta} cdc25-22 leu1-32::leu1+-pin1+), Sp66 (wee1-50), Sp88 (pin1{Delta} wee1-50) and Sp153 (pin1{Delta} wee1-50 leu1-32::leu1+-pin1+) cells were streaked out on YES plates and cultured at 29°C (top left), 32°C (bottom left) and 36°C (bottom right) for 5 days to compare cell growth and colony formation. The top right panel shows the schematic diagram of how these strains were streaked out on a YES plate.

 


View larger version (43K):

[in a new window]
  		Fig. 6. Deletion of pin1+ causes increased sensitivity to cyclosporin A (CsA) and okadaic acid (OA). (A) Sp1 (wild-type), Sp3 (wild-type), Sp23 (pin1{Delta}) and Sp25 (pin1{Delta}) cells were streaked out on YES plates containing no CsA (top right), 10 µg/ml CsA (bottom left panel) or 60 µg/ml CsA (bottom right panel) followed by incubation at 32°C for 3 days. The top left panel shows the schematic diagram of how these strains were streaked out on a YES plate. (B) Sp1 (wild-type), Sp3 (wild-type), Sp23 (pin1{Delta}) and Sp25 (pin1{Delta}) cells were streaked out on YES plates containing no OA (left panel) or 5 µM OA (right panel) followed by incubation at 32°C for 3 days. The strains were arranged on the plates as in A.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2001