Analysis of deletion phenotypes and GFP fissions of 21 novel Saccharomycescerevisiae open reading frames

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests o f deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitot ic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew wi th reduced rates. Two genes (YNL223w and YNL225c) were identified to be req uired for sporulation. In addition we also performed green fluorescent prot ein (GFP) tagging for localization studies. GFP labelling indicated the spi ndle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visibl e in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal wa s 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 identifiab le 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 re mained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew onl y poorly on galactose, indicating a toxic effect of the overexpressed fusio n 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%) ca ses. Copyright (C) 2000 John Wiley & Sons, Ltd.