Multicopy GFP-fusion Library of Fission Yeast
GFP-fusion library of Schizosaccharomyces pombe
Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library.
Da-Qiao Ding, Yuki Tomita, Ayumu Yamamoto, Yuji Chikashige, Tokuko Haraguchi and Yasushi Hiraoka.
Genes Cells (2000) 5, 169-190.
Abstract
Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe.
We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5' end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49,845 transformants yielded 6,954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products.
A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells.
Summary table
Localization Pattern
- Nuclear region
- general
- nuclear dots
- nucleolar region
- nuclear rim