Evidence for variation in the number of functional gene copies at the AmaR locus in chinese hamster cell lines

The hypothesis of functional hemizygosity has been examined for the alpha-amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, DHW, M3- 1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards alpha-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to alpha-amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3- 1 cell lines. The remaining activity in the latter cell lines shows alpha-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by alpha-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.