Synthesis of the tumour antigen and the major capsid protein of simian virus 40 in a cell-free system derived from Escherichia coli

Abstract

A coupled system for transcription and translation derived from Escherichia coli was programmed with the DNA of simian tumour virus 40 (SV40). SV40 DNA is an effective template in this bacterial coupled system. The products of reactions run in the presence of [ 35 S]methionine were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, by indirect immunoprecipitation and by two-dimensional tryptic fingerprinting. The results were compared with those of similar analyses of SV40-specific tumour antigen (T antigen) isolated from a monkey kidney cell line and primary mouse kidney cells infected with SV40 (Ahmad-Zadeh et al. , 1976), and with the SV40 major capsid protein VP1. It is concluded that most or all of VP1 and T antigen are coded by SV 40 DNA. Intact VP1 is made in vitro with low efficiency. Intact T antigen (molecular weight=86,000) is not made. The largest fragment of T antigen made in vitro has a molecular weight of 59,000. A physical mapping technique is used to locate the T antigen gene on SV40 DNA. SV40 DNA fragments generated by treatment with restriction endonucleases were individually concatamerized by treatment with bacteriophage T4 DNA ligase and used to programme protein synthesis in vitro . We confirm that T antigen is coded by the early region of SV40 DNA and, therefore, that part of T antigen must correspond to the region of the SV40 genetic map defined by ts A mutations.