Genetics of Human Cell Lines I. 8-Azaguanine Resistance, a Selective "Single-Step" Marker.

Abstract

A cloned wild-type population (strain D98S) derived from human bone marrow cells, which is inhibited by 8-azaguanine (AG) in concentrations in excess of 0.1 ug/ml, contains approximately 1 to 2% cells resistant to AG in 100-fold higher concentrations. A single-step mutational process (in broad meaning of the word) appears to be involved. The AG-resistant lines (D98/AG) are stable upon prolonged subculture either in presence or absence of drug. Mutation rate from AG sensitivity to resistance is of the order of 5 x 10-4/cell/ generation. Strain D98/AG, which exhibits only 3-fold increase in 8-azaguanosine (AGR) resistance over D98S line, contains approximately 0.01% mutants (D98/AGR) resistant in a single mutational step over 100-fold higher concentration of AGR. AG resistance and especially AGR resistance, are useful genetic markers, since at selective AG or AGR concentrations (a wide plateau at 1-10 ug/ml and 3-30 ug/ml respectively) colony formation by resistant cells is not impaired and sensitive cells are destroyed. A method for protein determination is described, based on colorimetric measurements of bromphenol blue eluted from HgCl2-fixed, glass -attached cells.