Over-expression

F-adjacent Transcript-10 (FAT 10) is composed of two ubiquitin-like domains and capable to mark conjugated proteins for proteasomal degradation independent of ubiquitin. FAT10 is inducible by IFN-y and TNF and induces apoptosis when over expressed. 

S100A1 is the most abundant in the myocardium but is also expressed in brain and other tissues. S100A1 was found to stimulate Ca2+-induced Ca2+-release (CICR) in skeletal muscle terminal cisternae. In the presence of nanomolar Ca2+-concentrations, S100A1 binds to the ryanodine receptor increasing its channel open probability, and was shown to enhance SR Ca2+-release and contractile performance. Several animal models (over expressing S100A1 or S100A1-deficient mice) have... 

OTRs are mainly expressed in myoepithelial cells of the galactiferus channels and the myometrium. The OTRs in vascular endothelial cells, renal epithelial cells (macula densa, proximal tubule) and cardiomyocytes induce the production of NO (vasodilation), natriuresis and release of ANP, respectively. The endometrium, ovary, amnion, testis, epididymis, prostate and thymus also express the OTR supporting a paracrine role of this peptide. Osteoblasts, osteoclasts, pancreatic islets cells, adipocytes, and several types of cancer cells also express OTRs. More over, expression of the OTR... 

Just as myosins are able to move along microfilaments, there are motor proteins that move along microtubules. Microtubules, like microfilaments, are polar polymeric assemblies, but unlike actin-myosin interactions, microtubule-based motors exist that move along microtubules in either direction. A constant traffic of vesicles and organelles is visible in cultured cells especially using time-lapse photography. The larger part of this movement takes place on micrombules and is stimulated by phorbol ester (an activator of protein kinase C), and over-expression of N-J aj oncoprotein (Alexandrova et al., 1993). 

Yan J. Wang J. Tissue D. Holaday A. S. Allen R. Zhang H. (2003) Protection of photosynthesis and seed production under water-deficit conditions in transgenic tobacco plants that over-express Arabidopsis ascorbate peroxidase // Grop Sci. V. 43. P. 1477-483. 

Clone Over-express Purify Produce crystals... 

Figure 2.4 Noradrenergic inhibition of Ca " currents and transmitter release in sympathetic neurons and their processes, (a) Inhibition of currents through N-type Ca " channels by external application of noradrenaline (NA) or by over-expression of G-protein P y2 subunits, recorded from the soma and dendrite of a dissociated rat superior cervical sympathetic neuron. Currents were evoked by two successive 10 ms steps from —70 mV to OmV, separated by a prepulse to -1-90 mV. Note that the transient inhibition produced by NA (mediated by the G-protein Go) and the tonic inhibition produced by the G-protein Piy2 subunits were temporarily reversed by the -1-90 mV depolarisation. (Adapted from Fig. 4 in Delmas, P et al. (2000) Nat. Neurosci. 3 670-678. Reproduced with permission), (b) Inhibition of noradrenaline release from neurites of rat superior cervical sympathetic neurons by the 2-adrenoceptor stimulant UK-14,304, recorded amperometrically. Note that pretreatment with Pertussis toxin (PTX), which prevents coupling of the adrenoceptor to Gq, abolished inhibition. (Adapted from Fig. 3 in Koh, D-S and Hille, B (1997) Proc. Natl. Acad. Sci. USA 1506-1511. Reproduced with permission)...

In addition to proteases, other inhibitors reduce the activity of amylase and other digestive enzymes (Ishimoto et al, 1999). Many varieties of beans produce a glycoprotein that complexes with and inhibits a-amylase (Mirkov et al, 1995). The amylase inhibitors are non-competitive and thermostable (Gallaher and Schneeman, 1986) and, unlike protease inhibitors, do not elicit heightened secretion of amylase (Toskes, 1986). Although over-expression... 

HAGUE A, DIAZ G D, HICKS D J, KRAJEWSKI s, REED c, PARASKEVA c (1997) Blc-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells however over expression of blc-2 does not protect against bak-mediated apoptosis. Int. J. Cancer 72 898-905. 

Metabolic control analysis (MCA) assigns a flux control coefficient (FCC) to each step in the pathway and considers the sum of the coefficients. Competing pathway components may have negative FCCs. To measure FCCs, a variety of experimental techniques including radio isotopomers and pulse chase experiments are necessary in a tissue culture system. Perturbation of the system, for example, with over-expression of various genes can be applied iteratively to understand and optimize product accumulation. 

The food technologist may be especially interested in the fate of the carotenoids in the seed oil. Like red palm oil, the resulting carotenoid-pigmented canola oil may be more stable due to the antioxidant properties of carotenoids and may be more attractive to consumers. Alternatively, for food security concerns, transgenic soybean or canola oils and seed meals that are genetically modified for more efficient bio-diesel production may be bio-safety marked with lipid-soluble carotenoids and water-soluble anthocyanins, respectively. Potatoes are excellent potential sources of dietary carotenoids, and over-expression of CrtB in tubers led to the accumulation of P-carotene. Potatoes normally have low levels of leaf-type carotenoids, like canola cotyledons. 

Over-expression of bacterial phytoene synthase led to only modest increases in pigment accumulation (except in the case of chloroplast-contaiifing tissues). Attention turned to CrtI, one gene that might control flux through the entire four desaturation steps from phytoene to lycopene (discussed in Section 5.3.2.4). Only a modest increase in carotenoid content in tomatoes and a variety of changes in carotenoid composition including more P-carotene, accompanied by an overall decrease in total carotenoid content (no lycopene increase), resulted when CrtI was over-expressed under control of CaMV 35S. Apparently, the bacterial desaturase... 

Plant phytoene synthase (Psy) has been used in a variety of transgenics. As noted above, P yl over-expression under a strong constitutive promoter caused a decrease in carotenoid accumulation, probably due to transcription silencing. Similarly, over-expression of the gene sequence backward (antisense) also silenced activity. In another approach to over-expression of tomato 1 in fruits, a synthetic alternative in which the third position of each codon was changed in order to avoid transcriptional silencing was successful in conditioning an increase in carotenoid accumulation. 

In rice endosperm that accumulated phytoene by virtue of daffodil Psy transgenesis, Burkhart et al. reported the failure of daffodil Pds over-expression in rice endosperm to condition accumulation of -carotene, even though an increase in PDS antigen was detected. ... 

The availability of precursor IPP may ultimately be most influential over accumulation of carotenoid metabolites. While over-expression of DXS and DXR in color complementation systems leads to hyperaccumulation of carotenoids (discussed in Section 5.3.3.3), over-expression of plant Dxs genes has not always been effective. Over-expression of DXS resulted in increased carotenoid accumulation in transgenic tomato and Arabidopsis, but over-expression of daffodil DXS in rice endosperm did not increase pigment accumulation. ... 

Over-expression and anti-sense constructs of LCYB have been tested in rice and tomato. In Golden Rice, daffodil LCYB was over-expressed but found to be unnecessary for accumulation of carotenes. Only PSY and CRTI are needed to accumulate carotenes and xanthophylls in the endosperm. 

Fortuitously, the bacterial gene product, CRTI, produces di -trans carotenoids and satisfies the stereo-chemical specificity of LYC B for all-trani substrates while also catalyzing the four desaturation steps from phytoene to lycopene. Nevertheless, over-expression of Crtl has been shown to have only a modest effect (two- to fourfold increases in tomatoes and carrots) in increasing flux through the pathway and some unexpected pleiotropic influences on activities upstream and downstream of the desaturations (reviewed by Fraser and Bramley and Giuliano °). 

Careful empirical selection of the expression platform for carotenogenesis has included selection of the best strains for stability and degree of accumulation and the selection of compatible drug-resistance combinations and low copy number polycistronic plasmids to enhance product accumulation by decrease of metabolic burden." 5 Matthews and Wurtzel discussed culture and induction conditions - that have been explored in most studies. Most efforts to engineer carotenoid biosynthesis in E. coli focused on the genes and enzymes of the pathway and had a modest effect on improved accumulation. For example, substitution and over-expression of a GGPPS that uses IPP directly (discussed in... 

FIGURE 5.3.5 Enhancement of lycopene accumulation in . coZi by over-expression of DXS. Lycopene accumulation (left) is enhanced (right) when E. coli cells carrying a carotenoid pathway gene cassette (+EIB) are further transformed with a dxs gene on a multicopy plasmid (+E1B +dxs). Lycopene hyperaccumulation was demonstrated by Matthews and Wurtzel. ... 

Section 5.3.2.3) combined with over-expression of IPPI resulted in enhanced astaxanthin accnmnlation to 1.4 mg/g dry cell weight (DCW). Further increases to 45 mg/g DCW were obtained by random mutagenesis of GGPPS, perhaps by altering enzyme response to substrate-level feedback inhibition. 

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