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)

Phage T7 RNA polymerase has been used extensively in Escherichia coli for high-level expression of selected genes placed under the control of the phage T7 gene 10 promoter. We have constructed an analogous system for use in plastids of higher plants. A T7 RNA polymerase chimeric gene containing a cauliflower mosaic virus 35S promoter and a tobacco ribulose-bisphosphate carboxylase/oxygenase small-subunit chloroplast transit-peptide sequence was introduced into tobacco by nuclear transformation. Stable plastid transformation of tobacco expressing the T7 RNA polymerase activity with a T7 promoter/beta-glucuronidase (GUS) reporter gene construct resulted in expression of GUS mRNA and enzyme activity in all tissues examined. Expression of GUS activity was extremely high in mature leaves, moderate in young leaves and petals, and low in stems, roots, and developing seeds. Plastid transformation of wild-type tobacco with the same chimeric GUS gene resulted in undetectable levels of GUS mRNA and enzyme activity. Genetic crosses demonstrated that a silent T7/GUS reporter gene could be activated in the F1 generation by transmission of an active nuclear T7 RNA polymerase gene from the male parent.
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PMID:Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. 804 84

Deletions in the spinach rubisco activase (Rca) promoter in transgenic tobacco were analyzed to define the regions necessary for conferring light-inducible and tissue-specific expression. Transgenic plants were constructed with Bal 31 deletions of the Rca promoter fused to the coding region of the bacterial reporter gene beta-glucuronidase (GUS). Analysis of the Rca deletion mutants localized the region conferring normal expression downstream from -294 relative to the Rca transcription start site. A second set of transgenic plants containing the cauliflower mosaic virus (CaMV) 35S enhancer fused to the 3' end of the Rca/GUS constructs demonstrated the presence of a light-responsive element between -150 and -78 active in leaves. Regions 10 bp long within the light-responsive region, which included putative G box and GT elements, were removed by recombinant polymerase chain reaction. Deletion of the G box element resulted in a loss of gene expression in the leaves of transgenic tobacco, while deletion of the GT motif caused a 10-100-fold increase in expression in roots. However, site-directed mutagenesis of the GT motif resulted in expression patterns identical to the normal promoter. These experiments demonstrated that light-inducible and tissue-specific expression of the Rca promoter involves multiple cis elements proximal to the transcription start site, and that interactions between these elements are essential for regulating expression.
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PMID:Localization of light-inducible and tissue-specific regions of the spinach ribulose bisphosphate carboxylase/oxygenase (rubisco) activase promoter in transgenic tobacco plants. 829 78

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding beta-glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-specific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.
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PMID:The promoter of rbcS in a C3 plant (rice) directs organ-specific, light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression. 1109 84

Expression of rbcS genes encoding small subunit of rubisco, most abundant protein in green tissue, is regulated by at least three parameters--tissue type, light conditions and stage of development. One of the green tissue-specific promoters of rbcS gene family was isolated from pigeonpea by PCR. Expression of uidA gene encoding beta-glucuronidase in the transgenic tobacco plants under the control of pigeonpea rbcS promoter, clearly showed that this promoter was as strong as pea rbcS3A promoter characterized earlier. Study of the sequence similarity with pea rbcS3A promoter, especially the region (boxes I and III) that is required for rbcS3A expression, showed more than 50% divergence. In contrast, pigeonpea promoter sequence isolated in the present study was more similar to that of spinach and rice rbcS promoters.
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PMID:Isolation and characterization of a green tissue-specific promoter from pigeonpea [Cajanus cajan (L.) Millsp.]. 1587 23

Tobacco has been studied as a possible host for the production of recombinant proteins. In this report, recombinant beta-glucuronidase (rGUS) was used as a model protein to study the feasibility of using polyethyleneimine (PEI) precipitation to fractionate acidic recombinant proteins from transgenic tobacco. Results showed that rGUS was preferentially precipitated when the PEI dosage was beyond 200mg PEI/g total protein. At 700-800 mg PEI/g total protein, nearly 100% rGUS was precipitated with less than 40% native tobacco proteins co-precipitated. Approximately 85-90% of the rGUS activity could be recovered from the precipitation pellet for an enrichment ratio of 2.7-3.4. As a comparison, anion exchange chromatography (AEX) yielded similar results to PEI precipitation with 66-90% rGUS activity recovered and an enrichment ratio of 1.8-3.1. The rGUS was further purified by an additional hydrophobic interaction chromatographic (HIC) step after precipitation or AEX. Similar results in terms of rGUS activity recovered and enrichment were obtained. The major interfering protein at the end of all purification steps is most likely the Fraction 1 protein ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). The presence of this protein is likely the cause for the varying and somewhat low enrichment ratios, but it may be later removed via a size-exclusion chromatography step. PEI precipitation offers the advantage of allowing significant sample concentration prior to subsequent purification techniques such as chromatography and is much less expensive than chromatographic methods as well. Through direct comparison, this study shows that PEI may be used as an initial fractionation step in replacement of AEX to facilitate the purification of acidic recombinant proteins from transgenic tobacco.
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PMID:Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta-glucuronidase from transgenic tobacco extract. 1695 Mar 25

Strong, tissue-specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a 'repressor-operator gene complex' (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root-specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn, LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using beta-glucuronidase (GUS) and mercuric ion reductase (merA) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root-specific tobacco RB7 promoter is demonstrated.
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PMID:Engineering a root-specific, repressor-operator gene complex. 1714 28