Chemical Mutagenesis at the Phosphoribosyltransferase Locus in Cultured Human Lymphoblasts

Abstract

The presence of selectable genetic markers in long-term human lymphoblast cultures would facilitate cell hybridization experiments on the biosynthesis of immunoglobulins, as well as other studies. This work reports the induction with ethylmethane sulfonate of 6-thioguanine -resistant, phosphoribosyltransferase -deficient mutants in a lymphoblast line from a patient with infectious mononucleosis. These cells were unusually sensitive, with a Do value of 28 sg of ethylmethane sulfonate per ml; the sensitivity curve followed a biphasic pattern suggesting the presence of 3% resistant cells. Ethylmethane sulfonate increased the frequency of mutants resistant to 6-thioguanine over 100-fold, to about 2 X 10-4; nitrosoguanidine was less effective. Almost all the mutants contained considerably less than 1% of the hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity of wild-type cells. The mutation did not appear to result from loss of an X chromosome.

Determination of DNA sequence changes induced by ethyl methanesulfonate in human cells, using a shuttle vector system.

“…Here we used the system to examine the mutagenic specificity of a member of another major group of mutagens, the alkylating agents. We chose ethyl methanesulfonate (EMS; Sigma Chemical Co.) for this study because it has been widely used as a mutagen in mammalian cells (2,3,9,11,24). Furthermore, because the mutagenic specificity of EMS has been determined for lacd in E. coli (5), we could directly compare the mutagenic effects of this alkylating agent in bacterial and human cells.…”

“…The mutations were obtained by using a shuttle vector system with the bacterial lacl gene as the target. Of the 54 mutations obtained, 53 were G:C to A:T transitions.Recent advances in molecular biology have led to the development of recombinant DNA vectors designed to provide information about the nature of the mutations induced by various mutagens and carcinogens in mammalian cells (1,24,15,20,21,25). These shuttle vector systems comprise plasmids with the ability to replicate in both mammalian and bacterial cells and with genes in which mutations can be scored in Escherichia coli.…”

Mutation studies with human fibroblasts.

“…Both types of cells may have much to offer in mutagenicity testing. At this time, lymphoblastoid lines exhibit great variability in cloning efficiencies and in sensitivities to the lethal and mutagenic effects of chemical mutagens (10). Their indefinitely prolonged in vitro propagability may depend on the presence of a viral (Epstein-Barr) genome, which might conceivably affect the mutational behavior of the cells.…”

Enrichment and Expansion of Specific Antibodyforming Cells by Adoptive Transfer and Clustering, and Their Use in Hybridoma Production

“…The process was performed at 37° with continual stirring. After fusion the cells were resuspended in RPMI-1640 with 10% FCS and HATD (Sato, Slesinski and Littlefield, 1972) for culture at 37° and 10% CO2 in air. Feeder cells were added to all cultures so that total spleen cell numbers in all three cultures would be equivalent and cultures distributed at 1-2 X 100 cells/ml into V-bottomed 96-well microtitre trays (Cooke, Microtitre).…”