Embryo protein extract

Figure 4. Alfalfa somatic embryo proteins separated after extraction by sucrose density gradient separation (left side of chromatogram) and SDS-polyacrylamide electrophoresis (right side of chromatogram). The 2,4-D-treated embryos express more IIS protein as shown in the sucrose gradient. The IIS protein has polypeptides that co-migrate with IIS seed protein as shown by SDS-PAGE. (Reproduced with permission from reference 4. Copyright 1985, Plenum Publishing.)...

Fig. 1. Immunological characterization of the 60 kDa biotinylated protein of carrot somatic embryos. Crude extract from carrot somatic embryos was subjected to SDS-PAGE and western analysis. The blots were probed with 1, 125i streptavidin 2, anti-60 kDa biotinylated protein 3, anti-30 kDa biotinylated protein. In both immunological westerns antibody-antigen complexes were detected with 25i Protein A.

Fig. 4. Electrophoretic (acrylamide gel) profiles of radioactivity of proteins extracted from Strongyhcentrotus purpuratus embryos after incorporation of l-valine- C at (I) 3-4 hours and at (II) 23-24 hours after fertilization, cultured with (lA and IIA) and without (IC and IIC) actinomycin D (20 /ig/ml) protein content per 0.25 ml sample for lA, IC, IIA, and IIC = 0.40, 0.35, 0.26, and 0.46 mg, respectively total radioactivity in protein per applied sample for lA, IC, IIA, and IIC = 39,800, 38,200, 22,275, and 15,900 cpm, respectively. Numbers (1-7) indicate repeatedly identifiable peaks. From Spiegel et al. (1965).

Aprotinin Maize ubiquitin promoter/ pinll terminator Maize ubiquitin 5 intron, barley a-amylase LP Extracellular matrix of embryo (maize seeds) In T5 0.35% of extractable seed proteins 16... 

Beside this dermatoxic activity pederin (147) has various biological activities (92). When administered in appropriate doses to partially hepatectomized rats, this compound stimulates development of hepatic tissues. The inhibitory effect at the cellular level has been found in chicken heart fibroblast cultures, and mice embryo, dog kidney, HeLa, and KB cell lines. In plants, root growth of Lupinus albus is inhibited and mitosis in Allium cepa blocked at the metaphasic stage. Also, pederin (147) inhibits protein synthesis and growth of yeast cells. In addition, the treatment of rat ascites sarcoma with purified extracts of P. fuscipes produces almost complete regression. 

Enzymic transfer of D-xylose from uridine 5 -(D-xylopyranosyI-HC pyrophosphate) to L-serine residues of endogenous protein acceptors from (a) a cell tumor of the mouse188 and (b) chick-embryo cartilage189 occurs in cell-free extracts of both of these tissues, in the absence of biosynthesis of protein. The enzyme preparations employed were from the supernatant liquor, although activity was also present in the insoluble fractions. In these two types of tissue, the acceptors are heparin and chondroitin sulfate, respectively, but the presence of other D-xylose-containing glycoproteins in ascites fluid from... 

Key Words Cell-free protein synthesis pure embryo isolation wheat extract preparation transcription and translation reactions. 

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.

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. 2. Removal of tritin from embryos. Extracts were prepared from unwashed or washed embryos (A) and the depurination assay was performed (B). Translation mixtures prepared with the extract from unwashed embryos were incubated for 0, 1, 2, 3, 4 h (lanes 1-5, respectively) mixtures with washed embryos were incubated for 0, 2, 4 h (lanes 10-12, respectively). Isolated RNA was treated with acid/aniline, and then separated on 4.5% polyacrylamide gels. Additionally, RNA was directly extracted from embryos with guanidine isothiocyanate-phenol and analyzed before (lane 7) and after (lane 8) treatment with acid/aniline. For the fragment marker, incubation was carried out in the presence of gypsophilin, a highly active ribosome-inactivating protein from Gypsophila elegance the arrow indicates the aniline-induced fragment.

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.

Singer and colleagues (68, 69) have successfully visualized cytoskeletal mRNAs and their associate proteins using biotinylated cDNA probes followed by antibodies to biotin and collodial gold-conjugated antibodies. Their method used in situ hybridization followed by whole mount TEM of Triton-extracted chicken embryo fibroblasts. Cytoskeletal mRNAs were found in close proximity to actin protein and further from tubulin filaments. While the whole mount technology does limit the technique to extracted cells, applications to thin sections will allow greater resolution. 

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. 

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