Wheat embryo

Miyamoto et al. (101) obtained four fractions from the seed coats of wheat which inhibited development of the wheat embryo. A component from one of the fractions responsible for 20% of the total inhibitor activity was crystallized but not identified. 

Table 15.1 The influence of zearalenone on the generative development of winter wheat cv. Grana after various periods of vernalization (According to Biesaga-Koscielniak 1998, modified). Isolated wheat embryos were cultured in sterile conditions on Murashige and Skoog (1962) media supplemented with 0 (control), 0.25, 0.50, 0.75 and 2.00 mg/dm3 of zearalenone during 14, 28 and 42 days at 5°C (vernalization). After these periods, plants were transferred to soil and cultivated at 20/17°C. Number of headed plants, generative development of apexes and number of vegetative ones was obtained after 100 days of grown at 20/17°C...

Jinsart, W. Ternai, B. Polya, G.M. Inhibition of wheat embryo calcium-dependent protein kinase and avian myosin light chain kinase by flavonoids and related compounds. Biol. Chem. Hoppe-Seyler, 372, 819-827 (1991)... 

Williamson, J.D. Quatrano, R.S. (1988). ABA regulation of two classes of embryo-specific sequences in mature wheat embryos. Plant Physiology 86, 208-15. 

This chapter contains protocols for high-throughput protein production based on the cell-free system prepared from eukaryote wheat embryos. 

Grind 5 g of isolated wheat embryo to a fine powder in liquid nitrogen. 

Prepare 50 pL of translational reaction mixture by combining 10 pL of wheat embryo extract (thus 20%), 2 pL of 1 mg/mL creatine kinase, 10 pL of 5X WEB, and 28 pL of dissolved mRNA from 25 pL of transcription mixture. 

Fig. 3. (Opposite page) A novel bilayer cell-free protein synthesis. (A) Schematic illustration of the method. Wheat embryo cell-free system as described under Subheading 4.2. (B) Synthesis of green fluorescent protein (GFP) by bilayer mode. (C) The bilayer method ( ), bilayer but mixed ( ). For the measurement of (14C)leucine incorporation, samples were vortexed and hot trichloroacetic acid-insoluble radioactivity in 5 pL in the batch reaction or 30 pL in the bilayer reaction, thus adjusted amount of extracts in each system. The inset shows autoradiograms, and arrowheads mark GFP. Reprinted from (16) by permission of Federation of the European Biochemical Societies.

Key Words Cell-free protein synthesis wheat embryo extract 5 and 3 ORF design pEU construction bilayer reaction method robotic automation. 

Preparation of a Highly Active and Robust Extract From Wheat Embryos... 

One of the most convenient and promising eukaryotic cell-free translation systems is based on wheat embryos, which store all the necessary components of translation. However, conventional wheat-germ extracts are plagued with a short half-life, and consequently, with low expression of proteins. In recent findings, we proposed that the possible cause for instability is the presence in the original endosperm extract of RNA (V-glycosidase. tritin, and possibly other... 

Fig. 3. Protein synthesis with an extract prepared from washed embryos. The batch system contains either 12 pL (24%) or 24 pL (48%) of extracts from washed (A) or unwashed wheat embryos (B). Protein synthesis was measured as hot tricholoroacetic acid insoluble radioactivity. Arrows show addition of substrates.

Fig. 9. Procedure for protein synthesis using the wheat embryo cell-free system. WEX is an abbreviation for wheat expression system.

Madin, K., Sawasaki, T., Ogasawara, T., and Endo, Y. (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Set. USA 97, 559-564. 

Johnston, F. B. and Stem, H. (1957) Mass isolation of viable wheat embryos. Nature 179, 160-161. 

Marcus, A. Efron, D. Weeks, D. P. The wheat embryo cell free system. In Methods in Enzymology, Volume 30. 

Bonen L, Cunningham RS, Gray MW, Doolittle WF (1977) Wheat embryo mitochondrial 18S ribosomal RNA Evidence for its prokaryotic nature. Nucleic Acids Res 4 663-667 Buchner P (1965) Endosymbiosis of animals with plant microorganisms. Interscience, New York... 

Vitamin C than strawberry, kiwi, orange, and tomato, and the fruit also contains a higher concentration of Vitamin E than wheat embryo, safflower, maize, and soybean (9). 

The same nucleic acid may be prepared from wheat embryo and was formerly called triticonucleic acid. A ribosenucleic acid from pancreas, which has been named allonucleic acid, has not been well characterized. 

Thioredoxin systems have also been demonstrated in L. leichmannii (39), rat Novikoff hepatoma (40), T4 phage infected E. coli (41, 42), yeast (43), rat liver (44), calf liver (45) and germinating wheat embryo (32). 

Robust and Cost-effective Cell-free Expression of Biopharmaceuticals Escherichia Coli and Wheat Embryo... 

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

As a result of those studies, Endo et al. developed the pEU series of vectors with various affinity tags for efficient expression in wheat embryo cell-free systems without the need for expensive cap analogues, pl-VEX WG (wheat germ) vectors are now commercially available (Roche) which similarly rely on translation-enhancing untranslated regions flanking the multi-cloning site. 

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