Genetics of somatic mammalian cells. V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants.

Abstract

The preceding paper of this series described genetic studies on a Chinese hamster cell which exhibited a specific growth requirement for proline. The nutritional deficiency was shown to be a mutation causing lack of the enzyme converting glutamic acid to the 'y-semialdehyde. The character was demonstrated to be stable, exhibiting a reversion frequency of 10-6 per generation as expected of a single gene mutation.1 The present study was undertaken in order to develop methods for the detection and isolation of additional nutritionally deficient mutants for the purpose of location of genes on chromosomes, measurement of linkage, and study of gene regulatory phenomena. The principle employed, which has been widely used in microbial genetics, involves placing a mixed cell population in a limited medium in which nutritionally deficient forms cannot grow, and applying an agent which will kill only dividing cells.2 Two agents were studied in these experiments. The first was H3-thymidine, which can readily be incorporated in lethal amounts into the DNA of mammalian cells.3 DNA synthesis has previously been shown to be dependent on protein synthesis.4 Experiments demonstrated that differential incorporation of H3-thymidine into proline-sufficient and -deficient cells could readily be achieved in proline-free media, with a consequent enrichment of the deficient mutanit. However, the large amounits of radioactivity required for the execution of such experiments on a routine basis made this method objectionable. Consequently, another approach was adopted, in which cells were incubated with 5-bromodeoxyuridine (BUdR) for a period sufficient to ensure its incorporation into DNA.5 Thereafter, the cells were exposed to light in the hope that those cells which had incorporated BUdR into their DNA would become light-sensitive in the or near-visible (sometimes referred to as visible) region and be killed as occurs in bacteriophage.6 Hence, if a mixed cell population is so treated in a minimal medium, onie may expect more BUdR incorporation and subsequent killing among the nutritionally sufficient cell population than among the deficient forms, which should be concentrated among the survivors. This paper describes experiments which quantitate the response of mammalian cells to BUdR followed by visible light and which also demonstrate that proline-defi