Use of an epitope-tagged cDNA library to isolate cDNAs encoding proteins with nuclear localization potential.
We have combined epitope tagging with an expression cDNA library in order to isolate cDNAs encoding nuclear proteins. This system allows us to detect proteins expressed from the cDNA library by using antibodies against the epitope tag. As a tag, we used the 85-aa N-terminal peptide of the SV40 T antigen which lacks the nuclear localization signal (NLS). A strong expression vector, pEF204 [Kim et al., Gene 91 (1990) 217-223], was modified into an epitope-tagging vector, pTkim, by putting the tag-coding region and a cDNA cloning site immediately after its promoter. From cDNA libraries constructed using pTkim, we isolated eight cDNA clones whose tagged proteins were localized within the nuclei. From partial sequence analysis, two cDNAs were shown to code for the ribosomal (r-) proteins, simian L44 and human L21, and the others were shown to be new. Furthermore, six cDNAs including those encoding the r-proteins could direct a non-karyophilic T antigen [Fischer-Fantuzzi et al., Virology 153 (1986) 87-95] into nuclei, showing that they have NLSs. These results indicate that this system is useful for isolating new cDNAs which code for nuclear proteins.
Sugano
S
S
Department of Virology, University of Tokyo, Japan.
Kim
D W
DW
Yu
Y S
YS
Mizushima-Sugano
J
J
Yoshitomo
K
K
Watanabe
S
S
Suzuki
F
F
Yamaguchi
N
N
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000276:immunology; D015183:Restriction Mapping; D017384:Sequence Deletion; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D000939:Epitopes; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011061:Poly A; D012313:RNA; D004247:DNA; D003513:Cycloheximide