Journal article
A proteomics approach to cloning fenestrin from the nuclear exchange junction of Tetrahymena
The Journal of eukaryotic microbiology, Vol.55(4), pp.245-256
07/2008
DOI: 10.1111/j.1550-7408.2008.00337.x
PMID: 18681839
Abstract
We set out to find the "fenestrin" gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila. First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector (R) software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.
Details
- Title: Subtitle
- A proteomics approach to cloning fenestrin from the nuclear exchange junction of Tetrahymena
- Creators
- Eric S. Cole - St. Olaf CollegePaul C. Anderson - St. Olaf CollegeRoss B. Fulton - St. Olaf CollegeMatthew E. Majerus - St. Olaf CollegeMegan G. Rooney - St. Olaf CollegeJohanna M. Savage - St. Olaf CollegeDouglas Chalker - Washington University in St. LouisJerry Honts - Drake UniversityMary E. Welch - St. Olaf CollegeAmy L. Wentland - St. Olaf CollegeErica Zweifel - St. Olaf CollegeDouglas J. Beussman - St. Olaf College
- Resource Type
- Journal article
- Publication Details
- The Journal of eukaryotic microbiology, Vol.55(4), pp.245-256
- DOI
- 10.1111/j.1550-7408.2008.00337.x
- PMID
- 18681839
- NLM abbreviation
- J Eukaryot Microbiol
- ISSN
- 1066-5234
- eISSN
- 1550-7408
- Publisher
- Wiley
- Number of pages
- 12
- Grant note
- R01 GM069593 / NIGMS NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS)
- Language
- English
- Date published
- 07/2008
- Academic Unit
- Pathology
- Record Identifier
- 9984966755802771
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