Characterization of the zinc finger protein encoded by the WT1 Wilms' tumor locus.
We analysed the biochemical properties of the transcription factor encoded by the putative tumor-suppressor gene present at the WT1 Wilms' tumor locus. A gene containing the full-length amino acid coding sequence of human wt1 was reconstructed from synthetic oligonucleotides and cloned into expression vectors for in vitro and in vivo protein synthesis. Polyclonal rabbit antibodies specific for the WT1 protein were raised to an Escherichia coli-produced 91 amino acid N-terminal segment and to a 136 amino acid C-terminal segment, which contains the zinc finger domain. WT1 produced by in vitro translation migrated as a 52 kDa protein on sodium dodecylsulfate-polyacrylamide gels and bound to the EGR consensus sequence in gel-retardation assays. Expression of the wt1 gene via transient transfection in COS-1 cells revealed a 52 kDa protein which was immunoprecipitated by both the N-terminal- and C-terminal-specific antisera. Immunofluorescence studies of wt1-transfected COS-1 cells revealed that the WT1 protein was localized to the nucleus. Metabolic labeling with [32P]orthophosphate failed to reveal significant phosphorylation of the WT1 protein in COS-1 cells. Two immunoreactive WT polypeptides of 52 and 54 kDa were observed in murine embryonic stem cells and COS-1 kidney cells and may represent previously identified splicing variants of WT1. These antisera should be useful in characterizing the structure and function of the WT1 protein in human Wilms' tumor specimens.
Morris
J F
JF
Wistar Institute, Philadelphia, Pennsylvania 19104.
Madden
S L
SL
Tournay
O E
OE
Cook
D M
DM
Sukhatme
V P
VP
Rauscher
F J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D016147:Genes, Tumor Suppressor; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D014162:Transfection; D009396:Wilms Tumor / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008715:Methionine