1. Aminopterin prevents the multiplication of HeLa cells in Eagle's medium. The joint addition of adenine and thymidine removes the inhibition.
2. The aminopterin-induced thymine deficiency specifically results in a cessation of cell division and a doubling in the average protein content of the cells. Continued starvation for thymine results in the inability of cells to proliferate after aminopterin is removed. After 6 days only 10–6 to 10–5 of the original population proliferates.
3. The omission of a single essential amino acid, such as arginine or glutamine, from the medium during deprivation of thymine prevents a net increase in protein and results in about a 105-fold greater cell survival after 6 days, when aminopterin is removed and the missing amino acid is supplied.
4. Glutamine-requiring, auxotrophic mutants of HeLa exhibit a high survival after exposure to aminopterin in a glutamine-free medium. Glutamine-independent HeLa cells show a much lower survival in the same medium. Low frequencies of the auxotrophs can be specifically and efficiently selected for in artificial mixtures of the two cell types by treatment with the glutamine-free medium containing aminopterin.