Isolation and cryopreservation of O-methylthreonine-resistant Rosa cell lines altered in the feedback sensitivity of l-threonine deaminase

Abstract

O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID50≃6·10(-6)M). Isoleucine is able to reverse efficiently and specifically this OMT toxicity. From OMT-resistant colonies occurring at a frequency of 1.58·10(-7) variants per cell plated at 10(-4)M OMT, the variant strains OMT(R)-1 and OMT(R)-2 were isolated, cloned via protoplasts and characterized. Both variants were ten times more resistant to OMT than the wildtype and were cross-resistant to another isoleucine analog, DL-4-thiaisoleucine. The resistant variants retained their resistance after storage for three years in liquid nitrogen. Both resistant strains were stable for several months when subcultured in the absence of OMT although it was shown in a reconstitution experiment that wildtype cells overgrow OMT(R)-2 variant cells if co-cultivated for many passages in drug-free medium. One case of instability was observed upon long-term subculturing in drug-free medium: the strain OMT(R)-1D(*) partially lost phenotypic properties. Resistance to OMT was followed qualitatively by a new method based on inhibition-zone formation in cell suspensions plated in agar medium. The OMT-resistant variants showed a reduction in sensitivity of the enzyme L-threonine deaminase to feedback inhibition by isoleucine, a decreased stability of L-threonine deaminase when stored at-18°C or incubated at +55°C and a two- to threefold increase of the free isoleucine pool within the cells. The genetical events and the biochemical mechanisms which might lead to the observed stable and biochemically defined character are discussed with particular reference to the high ploidy level of the Rosa cell line.