Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library

Background: Intracellular localization is an important part of the characte rization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluore scent protein (GFP)-fusion genomic DNA library of S. pombe. Results: We constructed the S. pombe GFP-fusion genomic DNA library by fusi ng, 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 p roteins would be under the control of the own promoters contained in the ge nomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 tr ansformants which exhibited GFP fluorescence, of which 728 transformants sh owed fluorescence localized to distinct intracellular structures such as th e nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localization s of the 250 GFP-fusion constructs was categorized as an image database; us ing this database, DNA sequences can be searched for based on the localizat ions of their products. Conclusions: 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 acti vities, which can be readily used for microscopic observation in living cel ls.