Analysis of a large-T-antigen variant expressed in simian virus 40-transformed mouse cell line mKS-A.
Earlier reports had suggested that the large T antigen expressed in simian virus 40 (SV40)-transformed mKS-A cells may be replication defective. Our experiments support these earlier observations showing that the mKS-A T antigen has a reduced DNA-unwinding activity in vitro. To investigate the molecular basis for this defect, we have isolated from an mKS-A genomic library an EMBL-3 bacteriophage clone carrying in its insert a full-length SV40 DNA element that most likely encodes the expressed T-antigen variant. DNA sequencing revealed only one nonconservative amino acid exchange, Asp to Asn at residue 636. Surprisingly, when a plasmid clone carrying the mKS-A T-antigen-coding sequence was transfected into monkey cells, we found that it replicated quite efficiently, probably suggesting that a high nuclear concentration of the variant T-antigen form compensates for the partial biochemical defect. However, a high nuclear concentration of T antigen was also found in mKS-A T-antigen-transformed mouse cells, yet a fusion of these cells to permissive monkey cells failed to induce in situ replication and excision of integrated SV40 DNA. We discuss possible reasons for the different behavior of T antigen in monkey cells and in mouse cells and suggest that one possibility for the replication-negative phenotype in transformed cells may be related to the fact that T antigen forms a tight complex with the cellular p53 protein in mouse cells but not in monkey cells.
Dora
S
S
Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Schwarz
C
C
Baack
M
M
Graessmann
A
A
Knippers
R
R
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D051379:Mice; D009154:Mutation; D010957:Plasmids; D011233:Precipitin Tests; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004265:DNA Helicases