P1104 : Expression of liver-specific cytochrome P450 isoenzymes and oxygenases in C3A cells prior to and after treatment with the ELAD liver support system

Journal of Hepatology · 2015-04-01 · 1 citations

Co-expression of the tobacco anthranilate synthase β subunit with its feedback-insensitive α subunit as a selectable marker that also markedly increases the free tryptophan content

In Vitro Cellular & Developmental Biology - Plant · 2015-08-26 · 3 citations

Expression of a feedback insensitive anthranilate synthase gene from tobacco increases free tryptophan in soybean plants

Plant Cell Reports · 2007-06-14 · 40 citations

Identification of Arabidopsis thaliana variants with differential glyphosate responses

Journal of Plant Physiology · 2006-10-31 · 18 citations

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation

Plant Cell Reports · 2004-09-15 · 22 citations

Use of the tobacco feedback-insensitive anthranilate synthase gene (ASA2) as a selectable marker for legume hairy root transformation

Plant Cell Reports · 2004-05-27 · 26 citations

Length of time in tissue culture can affect the selected glyphosate resistance mechanism

Planta · 2004-02-01 · 10 citations

Glyphosate selection of gene amplification in suspension cultures of 3 plant species

Physiologia Plantarum · 2001-08-01 · 65 citations

Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measured by the I50. The selection process required from 8 to 11 transfers to fresh medium over a total period from 161 to 312 days. The alfalfa and soybean lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. The tobacco lines had EPSPS enzyme activity levels more than 800-times higher than the original cultures. The EPSPS gene copy number and mRNA were increased in all of the lines as measured by southern and northern hybridization, respectively. Thus, as has been found before with most glyphosate-resistant suspension cultures, the resistance is caused by high EPSPS enzyme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gene amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphosate tolerance.

Targeting a Nuclear Anthranilate Synthase α-Subunit Gene to the Tobacco Plastid Genome Results in Enhanced Tryptophan Biosynthesis. Return of a Gene to Its Pre-Endosymbiotic Origin

PLANT PHYSIOLOGY · 2001-09-01 · 61 citations

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS alpha-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion at ndhF-trnL only expressed one size of the ASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS alpha-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.

Increasing Tryptophan Synthesis in a Forage Legume<i>Astragalus sinicus</i>by Expressing the Tobacco Feedback-Insensitive Anthranilate Synthase (ASA2) Gene

PLANT PHYSIOLOGY · 2000-07-01 · 77 citations

A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agrobacterium rhizogenes with kanamycin selection. The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 microM 5MT, whereas the controls were completely inhibited by 15 microM 5MT. Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 alpha-subunit can interact with the native A. sinicus beta-subunit to form an active enzyme. The ASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2 can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.