Gene mutation assays mutant selection

Another issue with gene mutation assays is how they allow mutant cells to be identified, visualized, and counted, in order to calculate the mutant frequency. Available methods for endogenous genes consist of using (1) a selective agent to select mutant cells in vitro after cell isolation and transfer to culture medium (e.g.,... 

The bacterial and mammalian cell assays for gene mutation were developed to measure statistically significant increases in the numbers of mutant colonies derived from rare events many millions of exposed cells must be plated out to allow the assessment of mutation frequency. The Salmonella typhimurium reverse mutation assay ( Ames test) is carried out in a variety of different mutant strains selected to identify the various classes of mutation. The test generates many hundreds of Petri dishes for counting and is not practical for profiling. 

CD-I mice were exposed to purified air or benzene by inhalation at 0.04, 0.1, or 1.0 ppm for 22 hours per day, 7 days per week for 6 weeks (Ward et al. 1992). The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period, lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The increased frequencies of mutant spleen lymphocytes were significant at the low and mid, but not the high dose, and the method does not take into account possible clonal expansion. Further evaluation of the induction of gene mutation at these dose levels seems warranted. 

Applicable to all assays that use X phage as shuttle vector (e.g., MutaTMMouse, and Big Blue mouse and rat), except gpt delta models. The XEGIO phage used as vector in the later model bears a mutation in the chiC gene, involved in positive mutant selection. 

In the second approach, herbicide-resistance mutations in the Arabidopsis ALS gene were studied in E. coli. To do this, wild type and mutant Arabidopsis genes were functionally expressed in E. coli, such that the plant genes complemented a branched chain amino acid auxotrophy in the bacteria (Smith et al. 1989, PNAS in press). ALS enzyme assays on extracts prepared from E. coli expressing the mutant Arabidopsis gene indicated that the mutant enzyme is resistant to sulfonylurea herbicides but is sensitive to the imidazolinone herbicide imazaquin. This selective... 

Plasmid harboring the mutated gene for Rubisco was used to transform E.coli cells. Rubisco was isolated from cells grown on 2YT medium under selective conditions, and purified as previously described (5). Analysis of the mutant protein involved analytical gel filtration, electron microcopy of negatively stained samples with uranyl acetate. The zymatic capability was assayed by RuBP dependent fixation of C02 (8). The extent of activation of the enzyme by CO and Mg, and the ability to bind phosphorylated ligands were tested using 2-carboxy arabinitol bisphosphate, CABP (9). 

---