Application of an immunoprecipitation procedure to the study of SV40 tumor antigen interaction with mouse genomic DNA sequences.
Simian Virus 40 (SV40) large T antigen is a DNA binding protein with high affinity for segments of the viral genome. To find out whether T antigen also binds to sequences of genomic cellular DNA we mixed T antigen and SAU 3 A restricted mouse DNA under stringent DNA binding conditions. Resulting protein-DNA complexes were immunoprecipitated using T antigen specific monoclonal or polyclonal antibodies. The DNA fragments in the immunoprecipitates were cloned in plasmid vectors. Four plasmid clones were selected for a detailed investigation of the inserted mouse DNA fragments. Nucleotide sequencing and DNase I footprint experiments showed that T antigen binds to sites in these fragments consisting of two tandemly oriented G(A)AGGC pentamers separated by AT rich spacers of different lengths. The cellular binding sites are very similar in their architecture to the SV40-DNA binding site I. The isolated cellular DNA fragments with T antigen binding sites occur only once or a few times in the mouse genome. Our data help to further define the structure of T antigen's DNA binding sites. The genetic functions of the isolated cellular DNA elements are not known.
Pollwein
P
P
Fakultät für Biologie, Universität Konstanz, FRG.
Wagner
S
S
Knippers
R
R
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis*; D001483:Base Sequence; D002461:Cell Line, Transformed; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011233:Precipitin Tests / Q000379:methods*
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D004247:DNA