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A Proteomics Approach to Cloning Fenestrin from the Nuclear Exchange Junction of Tetrahymena
Author(s) -
COLE ERIC S.,
ANDERSON PAUL C.,
FULTON ROSS B.,
MAJERUS MATTHEW E.,
ROONEY MEGAN G.,
SAVAGE JOHANNA M.,
CHALKER DOUGLAS,
HONTS JERRY,
WELCH MARY E.,
WENTLAND AMY L.,
ZWEIFEL ERICA,
BEUSSMAN DOUGLAS J.
Publication year - 2008
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2008.00337.x
Subject(s) - tetrahymena , biology , gene , cloning (programming) , microbiology and biotechnology , proteomics , nuclear protein , tetrahymena pyriformis , genetics , computer science , transcription factor , programming language
. 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 ® 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.