A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis

We describe here a selection strategy allowing the cloning of sequences tha t contain a functional nuclear targeting signal. Our method relies on the u se of green fluorescent protein fusion proteins to identify nuclear targeti ng sequences. Transfected cells expressing nuclear protein fusions were iso lated on the basis of their nuclear fluorescence using flow cytometry and t he transfected DNAs were recovered after bacterial transformation with tota l DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-f old enrichment in nuclear protein-encoding clones after a single round of s election. Among the 63 clones that have been partially sequenced to date, 2 5 (40%) corresponded to known nuclear proteins and 13 (20%) to previously u ncharacterized sequences. Despite their ability to target the green fluores cent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuc lear targeting sequences, which opens the way to a wide investigation of nu clear import mechanisms and to the identification of previously unknown nuc lear proteins, (C) 2000 Academic Press.