Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.
As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.
Simpson
J C
JC
Department of Cell Biology and Biophysics, EMBL Heidelberg, Germany.
Wellenreuther
R
R
Poustka
A
A
Pepperkok
R
R
Wiemann
S
S
D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics*; D019992:Databases as Topic; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D021381:Protein Transport; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins