ABSTRACT
In a lytic infection of a permissive host by SV40, the large tumor antigen (T antigen), which is a product of early transcription of the SV40 A gene, has been previously shown to autoregulate its own transcription by binding to SV40 DNA. The DNA region to which T antigen binds most tightly was synthesized and subsequently introduced into the bacterial plasmid pUC8. The interaction of SV40 T antigen with the DNA duplexes, derived from both chemical synthesis and the recombinant plasmid, were examined by using nitrocellulose filter binding assays. An SV40-adenovirus hybrid protein, AD2+D2 protein, was also tested. The SV40 T antigen was found to bind more tightly than the hybrid protein. Kinetic assays demonstrated that the association rates for the two proteins with the DNA binding site were equivalent; however, once formed, the T antigen-DNA complex dissociated more slowly than the AD2+D2 protein-DNA complex.
Subject(s)
Antigens, Viral, Tumor/genetics , DNA, Viral/metabolism , Simian virus 40/genetics , Base Sequence , Binding Sites , DNA, Recombinant/metabolism , Kinetics , Nucleic Acid Heteroduplexes , Oligodeoxyribonucleotides/metabolism , Phosphorylation , Simian virus 40/immunologyABSTRACT
We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides. It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone. Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable. Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications.
