• Journal of Plant Biotechnology
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• v.5 no.1
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• pp.33-41
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• 2003

Microspore-derived cell lines resistant to 5-methyltryptophan (5MT, a tryptophan analog) or S-(2-aminoethyl)-L-cysteine (AEC, a Iysine analog) were selected in rice by in vitro mutagenesis. For selection of 5MT or AEC resistant cell lines, suspension-cultured cells were irradiated with gamma rays. Thirteen 5MT resistant cell lines were selected and they were able to grow stably at 2 times higher 5MT concentration. A feedback insensitive form of anthranilate synthesis, the pathway specific control enzyme for tryptophan synthesis, was detected from the 5MT resistant lines. Contents of the free amino acids in five resistant lines (MR12-1 to MR12-5) showed a 7.4 to 46.6 times greater level than that in the control culture. Tryptophan, phenylalanine, and tyrosine levels in the shikimate pathway were 28.1 and 22.5 times higher in MR12-3 and MR12 4, respectively, than that measured in the control cells. Four AEC resistant cell lines were isolated from cultures grown on medium containing 1 mM AEC, They were able to grow stably with 2 mM AEC, while sensitive calli were inhibited by 0.5 mM AEC. Aspartate kinase activities of the resistant lines were insensitive to the natural inhibitor, Iysine, and accumulated 2.2 to 12.9-fold higher levels of free Iysine than that of the control cells. Especially, the levels of aspartate, asparagine, and methionine in the aspartate pathway showed higher accumulation in the AEC resistant lines than that in the control cells.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• IMMUNE NETWORK
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• v.5 no.4
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• pp.221-231
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• 2005

Background: Immune regulatory dendritic cells (DCs) play an important role in maintaining self-tolerance. Recent evidences demonstrate that DCs expressing indoleamine 2,3-dioxygenase (IDO), which is involved in tryptophan catabolism, play an important role in immunoregulation and tolerance and induce T cell apoptosis. This study was devised to examine the role of IDO in the oral tolerance induction in collagen-induced arthritis (CIA) mouse model. Methods: Beginning 2 weeks before immunization, CII was fed six times to DBA/1 mice and the effect on arthritis was assessed. In tolerized mice, $CD11c^+$ DCs were isolated and stimulated with CII, IFN-${\gamma}$, and LPS with or without IDO inhibitor, 1-methyl-DL-tryptophan (1-MT) and IDO expression by $CD11c^+$ DCs was analyzed using FACS and RT-PCR. The expression of IDO, MHC II, CD80, and CD86 by $CD11c^+$ DCs were examined using confocal microscopy. Regulatory effect of $CD11c^+$ DCs on Ag-specific T cell proliferative response to CII was examined by mixed lymphocyte reaction (MLR) with or without 1-MT. Results: The proportion of IDO-expressing $CD11c^+$ DCs was slightly higher in tolerized mice than in CIA mice and significantly increased after stimulation with CII, IFN-${\gamma}$, and LPS in an IDO-dependent manner. On confocal microscopic examination, the expression of IDO was higher and those of MHC II and CD86 were lower in CD11c + DCs from tolerized mice compared to those from CIA mice. On MLR, $CD11c^+$ DCs from tolerized mice inhibited T cell proliferative response to CII in an IDO-dependent manner. Conclusion: Enhanced IDO expression by $CD11c^+$ DCs from tolerized mice may contribute to the regulation of proliferative response of CII-reactive T cells and could be involved in the induction of oral tolerance to CII.