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

O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID₅₀6·10^-6M). 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^-4M 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.Abbreviations OMT l-O-methylthreonine - TD l-threonine deaminase     

Histidine-Mediated Control of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of the five tryptophan biosynthetic enzymes of Neurospora crassa was shown to be derepressed in histidine-starved cells. This histidine-mediated derepression was not due to a lowered intracellular concentration of tryptophan in these cells. Furthermore, histidine-mediated derepression of tryptophan enzymes was found to be coordinate and not subject to reversal by tryptophan of either exogenous or biosynthetic origin. The synthesis of tryptophan enzymes also was found to be coordinate in cells which were not histidine-starved. Although histidine is clearly involved in regulating the synthesis of tryptophan enzymes, it did not prevent either tryptophan-mediated derepression of tryptophan enzymes or indole-3-glycerol phosphate-mediated derepression of tryptophan synthetase.     

A growth regulatory loop that provides homeostasis to phytochrome a signaling

Phytochrome kinase substrate1 (PKS1) is a cytoplasmic protein that interacts physically with, and is phosphorylated by, the plant photoreceptor phytochrome. Here, we show that light transiently increases PKS1 mRNA levels and concentrates its expression to the elongation zone of the hypocotyl and root. This response is mediated by phytochrome A (phyA) acting in the very low fluence response (VLFR) mode. In the hypocotyl, PKS1 RNA and protein accumulation are maintained only under prolonged incubation in far-red light, the wavelength that most effectively activates phyA. Null mutants of PKS1 and its closest homolog, PKS2, show enhanced phyA-mediated VLFR. Notably, a pks1 pks2 double mutant has no phenotype, whereas overexpression of either PKS1 or PKS2 results in the same phenotype as the pks1 or pks2 single null mutant. We propose that PKS1 and PKS2 are involved in a growth regulatory loop that provides homeostasis to phyA signaling in the VLFR. In accordance with this idea, PKS1 effects are larger in the pks2 background (and vice versa). Moreover, the two proteins can interact with each other, and PKS2 negatively regulates PKS1 protein levels specifically under VLFR conditions.