Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.31 (beta-glucuronidase)
 7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of beta-glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.
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PMID:Optimization of delivery of foreign DNA into higher-plant chloroplasts. 210 74

The rumen anaerobic fungus Neocallimastix frontalis was biolistically transformed using plasmids containing the bacterial beta-glucuronidase gene (GUS) fused to the promoter sequences of the enolase gene from N. frontalis. Multiple copies of the plasmids were precipitated onto tungsten particles and delivered into zoosporangia and a mycelial mat by a helium-driven biolistic device. Transformants were detected by histochemical assay for beta-glucuronidase. It was found that the enolase promoter sequences tested were responsible for the transient expression of the beta-glucuronidase gene. This is the first study presenting results on the transformation of an anaerobic fungus.
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PMID:Transient expression of the beta-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis. 902 Nov 33

We show that the genetic transformation of Nicotiana tabacum can be achieved by bombarding intact cells and tissues with DNA-coated particles. Leaves or suspension culture cells were treated with tungsten microprojectiles carrying plasmid DNA containing a neomycin phosphotransferase gene. Callus harboring the foreign gene was recovered from the bombarded tissue by selection on medium containing kanamycin. Kanamycin-resistant plants have subsequently been regenerated from the callus derived from leaves. Transient expression of an introduced beta-glucuronidase gene was used to assess the efficiency of DNA delivery by microprojectiles. The frequency of cells that were stably transformed with the neomycin phosphotransferase gene was a few percent of the cells that transiently expressed the beta-glucuronidase gene. These results show that gene transfer by high-velocity microprojectiles is a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium.
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PMID:Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. 1659 93

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The beta-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 x 10(-3). In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals.
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PMID:Transient gene expression in maize, rice, and wheat cells using an airgun apparatus. 1666 78

A cell suspension culture of tobacco (Nicotiana tabacum L.) was used as a model to study injury to cells during biolistic transformation. Lawns of cells were bombarded with tungsten particles that were coated with a plasmid containing the beta-glucuronidase and the neomycin phosphotransferase II genes. When a gunpowder-driven biolistic device was used, numerous transiently expressing cells were focused around the epicenter of the blast which was manifested by a hole blown in the filter paper supporting the cells. However, transformed cells nearest the blast epicenter were injured and could not be recovered as stable transformants. The injury was primarily caused by physical trauma to the cells from gas blast and acoustic shock generated by the device. Postlaunch baffles or meshes placed in the gunpowder device reduced cell injury and increased the recovery of kanamycin-resistant colonies 3.5- and 2.5-fold, respectively. A newly developed helium-driven device was more gentle to the cells and also increased the number of transformants. Cell injury could be further moderated by using a mesh and a prelaunch baffle in the helium device. Toxicity of the tungsten microprojectiles also contributed to cell injury. Gold microprojectiles were not toxic and resulted in fourfold more kanamycin-resistant colonies than when similar quantities of similarly sized tungsten particles were used.
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PMID:Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. 1666 26