DNA TRANSFER OF BACTERIAL ASPARAGINE SYNTHETASE INTO MAMMALIAN CELLS

Publisher Summary This chapter presents a study in which DNA from a clone M13oriC81 containing bacterial asparagine synthetase is used to transform directly a rat cell line, Jensen sarcoma, to grow in asparagine-free medium containing B-aspartylhydroxamate. The efficiency of DNA transfer was about 2 to 20 colonies per 106 cells exposed per μg of DNA Southern blot analysis of the transformants showed that the M13oriC81 DNA was integrated into high molecular weight DNA. Unstable transformants that had lost their ability to grow in asparagine-free medium containing 3-aspartylhydroxamate also lost this M13oriC81 DNA. Transformants grown in increased amounts of 3-asparatylhydroxamate had increased amounts of M13oriC81 DNA. The resistance phenotype could be transferred using DNA from transformants and these second-step transformants also contained M13oriC81 DNA. Northern blot analysis showed that the M13oriC81 DNA, which was transcribed in RNA hybridized with M13oriC81 DNA, was detected in a polysome fraction obtained from transformants. Western blot analysis of two-dimensional gels of proteins of transformants using anti-asparagine synthetase antibody showed new spots with some of the properties of the bacterial enzyme. These results indicated that a bacterial gene can be introduced and expressed directly, if inefficiently, in mammalian cells.