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Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames
Author(s) -
Brachat Arndt,
Liebundguth Nicole,
Rebischung Corinne,
Lemire Sophie,
SchÄrer Florian,
Hoepfner Dominic,
Demchyshyn Vasyl,
Howald Isabelle,
Düsterhöft Andreas,
Möstl Dörte,
Pöhlmann Rainer,
Kötter Peter,
Hall Michael N.,
Wach Achim,
Philippsen Peter
Publication year - 2000
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(200002)16:3<241::aid-yea517>3.0.co;2-t
Subject(s) - green fluorescent protein , biology , orfs , open reading frame , mutant , fusion protein , gene , phenotype , nuclear localization sequence , protein subcellular localization prediction , microbiology and biotechnology , genetics , peptide sequence , recombinant dna
As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c–GFP) and the nucleus (Ynl075w–GFP) as the sites of action of two proteins. Ynl080c–GFP and Ynl081c–GFP fluorescence was visible in dot‐shaped and elongated structures, whereas the Ynl022c–GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C‐terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5′–GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP–Ynl020c and GFP–Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases. Copyright © 2000 John Wiley & Sons, Ltd.