High-affinity SV40 T-antigen binding sites in the human genome.
We describe two different approaches to isolate human genomic sequences possessing high-affinity binding sites for the simian virus 40 (SV40) large T antigen. First, SV40 T antigen was added to Sau3A-restricted human DNA; the resulting T-antigen-DNA complexes were collected after repeated passages through nitrocellulose filters. The second approach involves the specific immunoprecipitation of chromatin fragments, generated by Sau3A treatment of nuclear chromatin from SV40-transformed human cells. The DNA fragments obtained were cloned in plasmid vectors for further investigation. Using the filter binding approach we isolated four different fragments with high-affinity binding sites. The binding site in one fragment was related to the strong T-antigen binding site I in the SV40 genome. The other three fragments contained multiple recognition pentamers, GA(G)GGC. Only one fragment with a high-affinity binding site was identified among the immunoprecipitable chromatin fragments. This DNA fragment belongs to the L1 family of human repetitive DNA. We present evidence suggesting that a significant fraction of human L1 elements possesses T-antigen binding sites. L1-related sequences appear as extrachromosomal elements in an SV40-transformed human cell line, and the amount of extrachromosomal L1 DNA was found to increase after fusion of transformed cells to permissive monkey cells.
Gruss
C
C
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Wetzel
E
E
Baack
M
M
Mock
U
U
Knippers
R
R
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002472:Cell Transformation, Viral; D002843:Chromatin / Q000302:isolation & purification; D003001:Cloning, Molecular; D003852:Deoxyribonucleoproteins / Q000302:isolation & purification; D005374:Filtration; D006801:Humans; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid
D000952:Antigens, Polyomavirus Transforming; D002843:Chromatin; D003852:Deoxyribonucleoproteins