Transient expression of the β-glucuronidase gene in embryogenic callus of Picea mariana following microprojection

A microprojection protocol using the DuPont Biolistic particle delivery system and the -glucuronidase (GUS) reporter gene fused with the 35S promoter of Cauliflower mosaic virus (CaMV) was developed for Picea mariana callus. Comparison of four tungsten microprojectile sizes showed the highest transient gene expression with 1.11m diameter particles. Adsorption of DNA on the microcarriers using calcium chloride led to higher GUS gene activity than using polyethylene glycol. GUS gene activity in P. mariana was the highest when cells were treated 5 and 6 days after subculturing to fresh media. The wheat ABA-inducible Em gene promoter yielded 4.5 times higher GUS gene activity than the 35S CaMV promoter. Comparison of transient GUS gene expression among 10 P. mariana embryogenic cell lines from six different open-pollinated families showed comparable gene activity, with the exception of one family showing no GUS gene activity.     

Transient expression of the β-glucuronidase gene in tissues ofArabidopsis thaliana by bombardment-mediated transformation

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun.     

Transient expression of the β-glucuronidase gene introduced into barley coleoptile cells by microinjection

A -glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, -glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for -glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the -glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.     

Secondary embryogenesis and transient expression of the β-glucuronidase gene in hypocotyls of rapeseed microspore-derived embryos

Secondary embryogenesis from rapeseed microspore-derived embryos (MDEs) was studied in three Brassica napus L. cultivars Global, PF₇₀₄ and Option. The best results in terms of secondary embryogenesis percentage obtained in cultures of Global and PF₇₀₄ MDEs (75.88 and 65.97 %, respectively) and PF₇₀₄ produced the highest number of secondary embryos per each primary embryo (14.91 ± 2.18). After optimization of physical parameters, rapeseed hypocotyls of MDEs were bombarded with microcarriers coated with a plasmid containing GUS reporter gene. The highest levels of transient GUS expression were obtained using bombardment with gold particles of 1.6 μm, at helium pressure of 9.3 MPa, a bombardment distance of 9 cm, chamber vacuum pressure of 7.1 × 10⁻⁶ kPa and single bombardment in bombardment medium containing 0.4 M mannitol.     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

Golgi β-glucuronidase of androgen-stimulated mouse kidney

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.     

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.     

Tissue specific expression of β-glucuronidase gene driven by heterologous promoters in transgenic cauliflower plants

In this paper we compare five heterologous promoters fused to β-glucuronidase gene in their influence on localization of GUS activity in cauliflower (Brassica oleracea var. botrytis) tissues: roots, leaves, petioles and curds. A constitutive promoter CaMV 35S and four tissue specific promoters were used: extAP from rape, PsMTAP from pea, RBCS3CP from tomato and SRS1P from soybean, and introduced into cauliflower seedling explants using Agrobacterium rhizogenes mediated transformation. Quantitative and histochemical GUS assays confirmed tissue specific gus expression. It was found that extAP promoter was the most active in petioles but also caused a significant gus expression in curds. GUS activity was hardly observed in curd and restricted only to its epidermis when PsMTAP promoter drove the gene. RBCS3CP and SRS1P promoters controlled similar expression of the gus gene throughout the plant except for curd where RBCS3CP was almost inactive.     

Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the β-glucuronidase reporter gene

The Biolistic^® microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the -glucuronidase gene.Abbreviations AMV alfalfa mosaic virus - AMVE alfalfa mosaic virus translational enhancer - EM protein of mature wheat embryo - GUS P-glucuronidase gene - MUG 4-methylumbelliferyl -D-glucuronide - NPT II neomycin phosphotransferase - X-GLU 5-bromo-4-chloro-3-indolyl -D-glucuronic acid     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of -glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.     

Stimulation of hepatic microsomal β-glucuronidase by calcium

Hydrolysis of 3-methylumbelliferyl glucuronide by liver microsomal β-glucuronidase is enhanced about 2-fold by micromolar concentrations of Ca²⁺; half-maximal stimulation occurs with 0.35 μM Ca²⁺. Dissociation of the enzyme from microsomal membranes by various treatments increases basal β-glucuronidase activity and markedly decreases the sensitivity of the enzyme to Ca²⁺. Under similar conditions, the soluble lysosomal form of the enzyme is insensitive to Ca²⁺. Ca²⁺ stimulation was unaltered by addition of calmodulin inhibitors or exogenous calmodulin. Thus, interaction of cytosolic Ca²⁺ with membrane bound β-glucuronidase may modulate glucuronidation in intact hepatocytes via a novel, calmodulin-independent mechanism.     

Human β-glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids

-Glucuronidase (GUS) has become an important enzyme model for the genetic study of molecular disease, enzyme realization, and therapy, and for the biogenesis and function of the lysosome and lysosomal enzymes. The genetics of human -glucuronidase was investigated utilizing 188 primary man-mouse and man-Chinese hamster somatic cell hybrids segregating human chromosomes. Cell hybrids were derived from 16 different fusion experiments involving cells from ten different and unrelated individuals and six different rodent cell lines. The genetic relationship of GUS to 28 enzyme markers representing 19 linkage groups was determined, and chromosome studies on selected cell hybrids were performed. The evidence indicates that the -glucuronidase gene is assigned to chromosome 7 in man. Comparative linkage data in man and mouse indicate that the structural gene GUS is located in a region on chromosome 7 that has remained conserved during evolution. Involvement of other chromosomes whose genes may be important in the final expression of GUS was not observed. A tetrameric structure of human -glucuronidase was demonstrated by the formation of three heteropolymers migrating between the human and mouse molecular forms in chromosome 7 positive cell hybrids. Linkage of GUS to other lysosomal enzyme genes was investigated. -Hexosaminidase HEX _B) was assigned to chromosome 5; acid phosphatase₂ (ACP ₂) and esterase A₄ (ES-A ₄) were assigned to chromosome 11; HEX _A was not linked to GUS; and -galactosidase (-GAL) was localized on the X chromosome. These assignments are consistent with previous reports. Evidence was not obtained for a cluster of lysosomal enzyme structural genes. In demonstrating that GUS was not assigned to chromosome 9 utilizing an X/9 translocation segregating in cell hybrids, the gene coding for human adenylate kinase₁ was confirmed to be located on chromosome 9.Supported by NIH Grants HD 05196, GM 20454, and GM 06321, by NSF Grant BMS 73-07072, and by HEW Maternal and Child Health Service, Project 417.     

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

In Vitro Cellular & Developmental Biology - Plant - We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS)...     

Synthesis of indole analogs as potent β-glucuronidase inhibitors

Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against β-glucuronidase inhibitory is utmost essential. In this study indole analogs (1–35) were synthesized, characterized using various spectroscopic techniques including ¹H NMR and EI-MS and evaluated for their β-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC₅₀ values ranging between 0.50 and 53.40 μM, with reference to standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96 Å) and thus preventing Glu451 from functioning as proton donor residue.     

Synthesis of benzimidazole derivatives as potent β-glucuronidase inhibitors

Twenty five 4, 6-dichlorobenzimidazole derivatives (1–25) have been synthesized and evaluated against β-glucuronidase inhibitory activity. The compounds which actively inhibit β-glucuronidase activity have IC₅₀ values ranging between 4.48 and 46.12 μM and showing better than standard d-saccharic acid 1,4 lactone (IC₅₀ = 48.4 ± 1.25 μM). Molecular docking provided potential clues to identify interactions between the active molecules and the enzyme which further led us to identify plausible binding mode of all the benzimidazole derivatives. This study confirmed that presence of hydrophilic moieties is crucial to inhibit the human β-glucuronidase.